Compositions comprising recombinant bacillus cells and another biological control agent

ABSTRACT

The present invention relates to a composition comprising a) recombinant exosporium-producing  Bacillus  cells that express a fusion protein comprising: (i) at least one plant growth stimulating protein or peptide and (ii) a targeting sequence that localizes the fusion protein to the exosporium of the  Bacillus  cells; and b) at least one further biological control agent selected from particular microorganisms disclosed herein and/or a mutant of a specific strain of a microorganism disclosed herein having all identifying characteristics of the respective strain, and/or at least one metabolite produced by the respective strain, and/or at least one metabolite produced by the respective strain that exhibits activity against insects, mites, nematodes and/or phytopathogens in a synergistically effective amount. Furthermore, the present invention relates to the use of this composition as well as a method for enhancing plant growth, promoting plant health, and/or reducing overall damage of plants and plant parts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/051,911, filed Sep. 17, 2014, the content of which isincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII-formatted sequence listing with a file named“BCS149057WO_ST25.txt” created on Sep. 14, 2015, and having a size of152 kilobytes, and is filed concurrently with the specification. Thesequence listing contained in this ASCII-formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND Field of the Invention

The present invention relates to a composition comprising (i)recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (x) at least one plant growth stimulating protein orpeptide; and (y) a targeting sequence that localizes the fusion proteinto the exosporium of the Bacillus cells; and (ii) at least one furtherbiological control agent selected from particular microorganismsdisclosed herein and/or a mutant of a specific strain of a microorganismdisclosed herein having all identifying characteristics of therespective strain, and/or at least one metabolite produced by therespective strain that exhibits the ability to improve plant growthand/or health and/or activity against insects, mites, nematodes and/orphytopathogens in a synergistically effective amounts. Furthermore, thepresent invention relates to the use of this composition as well as amethod for enhancing plant growth, promoting plant health, and/orreducing overall damage of plants and plant parts.

Background of the Invention

In crop protection, there is a continuous need for applications thatimprove the health and/or the growth of plants. Healthier plantsgenerally result in higher yields and/or better quality of a plant orits products.

In order to promote plant health, fertilizers are employed worldwide,based on both inorganic and organic substances. A fertilizer may be asingle substance or a composition, and is used to provide nutrients toplants. A major breakthrough in the application of fertilizers was thedevelopment of nitrogen-based fertilizer by Justus von Liebig around1840. Fertilizers, however, can lead to soil acidification anddestabilization of nutrient balance in soil, including depletion ofminerals and enrichment of salt and heavy metals. In addition, excessivefertilizer use can lead to alteration of soil fauna as well ascontaminate surface water and ground water. Further, unhealthfulsubstances such as nitrate may become enriched in plants and fruits.

In addition, insecticides and fungicide are employed worldwide tocontrol pests. Synthetic insecticides or fungicides often arenon-specific and therefore can act on organisms other than the targetorganisms, including other naturally occurring beneficial organisms.Because of their chemical nature, they may also be toxic andnon-biodegradable. Consumers worldwide are increasingly conscious of thepotential environmental and health problems associated with theresiduals of chemicals, particularly in food products. This has resultedin growing consumer pressure to reduce the use or at least the quantityof chemical (i.e., synthetic) pesticides. Thus, there is a need tomanage food chain requirements while still allowing effective pestcontrol.

A further problem arising with the use of synthetic insecticides orfungicides is that the repeated and exclusive application of aninsecticide or fungicides often leads to selection of resistant animalpests or microorganisms. Normally, such strains are also cross-resistantagainst other active ingredients having the same mode of action. Aneffective control of the pathogens with said active compounds is thennot possible any longer. However, active ingredients having newmechanisms of action are difficult and expensive to develop.

The use of biological control agents (BCAs) is an alternative tofertilizers and synthetic pesticides. In some cases, the effectivenessof BCAs is not at the same level as for fertilizers or for conventionalinsecticides and fungicides, especially in case of severe infectionpressure. Consequently, in some circumstances, biological controlagents, their mutants and metabolites produced by them are, inparticular in low application rates, not entirely satisfactory. Thus,there is a constant need for developing new, alternative planthealth-enhancing and/or plant protection agents which in some areas atleast help to fulfill the above-mentioned requirements.

SUMMARY

In view of this, it was in particular an object of the present inventionto provide compositions which have an enhanced ability to improve plantgrowth and/or to enhance plant health or which exhibit enhanced activityagainst insects, mites, nematodes and/or phytopathogens.

Accordingly, it was found that these objectives are achieved with thecompositions according to the invention as defined in the following. Byapplying a) recombinant exosporium-producing Bacillus cells that expressa fusion protein comprising: (i) at least one plant growth stimulatingprotein or peptide selected from the group consisting of an enzymeinvolved in the production or activation of a plant growth stimulatingcompound; an enzyme that degrades or modifies a bacterial, fungal, orplant nutrient source; and a protein or peptide that protects a plantfrom a pathogen or a pest; and (ii) a targeting sequence that localizesthe fusion protein to the exosporium of the Bacillus cells; and b) atleast one particular strain disclosed herein other than said recombinantBacillus cells according to the invention, the ability to enhancepreferably in a superadditive manner (i) plant growth, plant yieldand/or plant health and/or (ii) the activity against insects, mites,nematodes and/or phytopathogens.

References herein to targeting sequences, exosporium proteins,exosporium protein fragments, fusion proteins, and recombinantexosporium producing Bacillus cells that express such fusion proteinsshould not be considered to be stand-alone embodiments. Instead,throughout the present application, references to the targetingsequences, exosporium proteins, exosporium protein fragments, fusionproteins, and recombinant exosporium producing Bacillus cells thatexpress such fusion proteins should be considered to be disclosed andclaimed only in combination (and preferably in a synergisticcombination) with one or more of the particular biological controlagents described herein. Furthermore, references to “the particularmicroorganisms disclosed herein” or “to the particular biologicalcontrol agents described, or disclosed, herein” are intended toencompass the biological control agents and the microorganisms,including the strains, mutants and metabolites thereof, as described inparagraphs [000183]-[000226] below.

The present invention is directed to a composition comprising a)recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide selected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound and anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource or a protein or peptide that protects a plant from a pathogen;and (ii) a targeting sequence that localizes the fusion protein to theexosporium of the Bacillus cells; and b) at least one further anddifferent particular biological control agent disclosed herein and/or amutant of a specific strain of a microorganism disclosed herein havingall identifying characteristics of the respective strain, and/or atleast one metabolite produced by the respective strain that exhibitsactivity against insects, mites, nematodes and/or phytopathogens insynergistically effective amounts.

In some embodiments, the targeting sequence comprises an amino acidsequence having at least about 43% identity with amino acids 20-35 ofSEQ ID NO: 1, wherein the identity with amino acids 25-35 is at leastabout 54%; a targeting sequence comprising amino acids 1-35 of SEQ IDNO: 1; a targeting sequence comprising amino acids 20-35 of SEQ ID NO:1; a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1; atargeting sequence comprising amino acids 22-33 of SEQ ID NO: 1; atargeting sequence comprising amino acids 20-31 of SEQ ID NO: 1; atargeting sequence comprising SEQ ID NO: 1; or an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 2.

In other embodiments, the recombinant Bacillus cells are cells of aBacillus cereus family member such as Bacillus anthracis, Bacilluscereus, Bacillus thuringiensis, Bacillus mycoides, Bacilluspseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillusweihenstephensis, Bacillus toyoiensis and combinations thereof. In afurther embodiment, the recombinant Bacillus cells are cells of Bacillusthuringiensis BT013A.

In certain aspects, the fusion protein comprises an enzyme involved inthe production or activation of a plant growth stimulating compoundselected from the group consisting of an acetoin reductase, anindole-3-acetamide hydrolase, a tryptophan monooxygenase, anacetolactate synthetase, an α-acetolactate decarboxylase, a pyruvatedecarboxylase, a diacetyl reductase, a butanediol dehydrogenase, anaminotransferase, a tryptophan decarboxylase, an amine oxidase, anindole-3-pyruvate decarboxylase, an indole-3-acetaldehyde dehydrogenase,a tryptophan side chain oxidase, a nitrile hydrolase, a nitrilase, apeptidase, a protease, an adenosine phosphate isopentenyltransferase, aphosphatase, an adenosine kinase, an adenine phosphoribosyltransferase,CYP735A, a 5′ribonucleotide phosphohydrolase, an adenosine nucleosidase,a zeatin cis-trans isomerase, a zeatin O-glucosyltransferase, aβ-glucosidase, a cis-hydroxylase, a CK cis-hydroxylase, a CKN-glucosyltransferase, a 2,5-ribonucleotide phosphohydrolase, anadenosine nucleosidase, a purine nucleoside phosphorylase, a zeatinreductase, a hydroxylamine reductase, a 2-oxoglutarate dioxygenase, agibberellic 2B/3B hydrolase, a gibberellin 3-oxidase, a gibberellin20-oxidase, a chitosanase, a chitinase, a β-1,3-glucanase, aβ-1,4-glucanase, a β-1,6-glucanase, an aminocyclopropane-1-carboxylicacid deaminase, and an enzyme involved in producing a nod factor.

In other aspects, the fusion protein comprises an enzyme that degradesor modifies a bacterial, fungal, or plant nutrient source selected fromthe group consisting of a cellulase, a lipase, a lignin oxidase, aprotease, a glycoside hydrolase, a phosphatase, a nitrogenase, anuclease, an amidase, a nitrate reductase, a nitrite reductase, anamylase, an ammonia oxidase, a ligninase, a glucosidase, aphospholipase, a phytase, a pectinase, a glucanase, a sulfatase, aurease, a xylanase, and a siderophore.

In some embodiments, the fusion protein is expressed under the controlof a sporulation promoter native to the targeting sequence, exosporiumprotein, or exosporium protein fragment of the fusion protein. Thefusion protein may be expressed under the control of a high-expressionsporulation promoter. In certain aspects, the high-expressionsporulation promoter comprises a sigma-K sporulation-specific polymerasepromoter sequence. In other aspects, the sporulation promoter comprisesa nucleic acid sequence having at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity, at least 96%identity, at least 97% identity, at least 98% identity, or at least 99%identity with a nucleic acid sequence of any one of SEQ ID NOS: 85-103.In some embodiments, the sporulation promoter comprises a nucleic acidsequence having 100% identity with a nucleic acid sequence of any one ofSEQ ID NOS: 85-103.

In other embodiments, the at least one biological control agent is aBacillus subtilis or Bacillus amyloliquefaciens strain that produces afengycin or plipastatin-type compound, an iturin-type compound, and/or asurfactin-type compound. For background, see the following reviewarticle: Ongena, M., et al., “Bacillus Lipopeptides: Versatile Weaponsfor Plant Disease Biocontrol,” Trends in Microbiology, Vol 16, No. 3,March 2008, pp. 115-125. Bacillus strains capable of producinglipopeptides include Bacillus subtilis QST713, Bacillusamyloliquefaciens strain D747 (available as BACSTAR® from Etec CropSolutions, NZ and also available as DOUBLE NICKEL™ from Certis, US);Bacillus subtilis MBI600 (available as SUBTILEX® from Becker Underwood,US EPA Reg. No. 71840-8); Bacillus subtilis Y1336 (available as BIOBAC®WP from Bion-Tech, Taiwan, registered as a biological fungicide inTaiwan under Registration Nos. 4764, 5454, 5096 and 5277); Bacillusamyloliquefaciens, in particular strain FZB42 (available as RHIZOVITAL®from ABiTEP, DE); and Bacillus subtilis var. amyloliquefaciens FZB24 isavailable from Novozymes Biologicals Inc. (Salem, Va.) or Syngenta CropProtection, LLC (Greensboro, N.C.) as the fungicide TAEGRO® or TAEGRO®ECO (EPA Registration No. 70127-5).

In still other embodiments, the at least one biological control agent isselected from the group consisting of Bacillus pumilus strain QST2808,Bacillus subtilis strain QST713, Bacillus subtilis strain QST30002,Bacillus subtilis strain QST30004, Streptomyces microflavus strain NRRLB-50550, Streptomyces microflavus strain M, Bacillus firmus strainI-1582, mutants thereof having all the identifying characteristics ofthe respective strains, and at least one metabolite produced by therespective strains that exhibits activity against insects, mites,nematodes and/or phytopathogens.

In some embodiments, the composition of the present invention comprisesa) recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide selected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound and anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource; or a protein or peptide that protects a plant from a pathogen;and (ii) a targeting sequence that localizes the fusion protein to theexosporium of the Bacillus cells; and b) Bacillus firmus strain I-1582in a synergistically effective amount.

In some embodiments, the composition of the present invention comprisesa) recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide selected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound and anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource or at least one protein or peptide that protects a plant from apathogen; and (ii) a targeting sequence that localizes the fusionprotein to the exosporium of the Bacillus cells; and b) Bacillussubtilis strain QST713 in a synergistically effective amount.

In some embodiments, the composition of the present invention comprisesa) recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide selected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound and anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource or at least one protein or peptide that protects a plant from apathogen; and (ii) a targeting sequence that localizes the fusionprotein to the exosporium of the Bacillus cells; and b) Bacillus pumilusstrain QST2808 in a synergistically effective amount.

In yet other embodiments, the composition further comprises c) at leastone fungicide and/or d) at least one insecticide. The at least onefungicide and/or the at least one insecticide may be synthetic. In oneaspect of such embodiment, the biological control agent and additionalinsecticide of such composition comprise Bacillus firmus strain I-1582and clothianidin, respectively.

In a particular aspect of the above embodiments (i) the biologicalcontrol agent is Bacillus subtilis QST713 or mutants having all theidentifying characteristics of the Bacillus subtilis QST713 and/or atleast 95% or at least 98% sequence identity to Bacillus subtilis QST713;(ii) the targeting sequence comprises an amino acid sequence having atleast about 43% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 54%; (iii) theplant growth stimulating protein or peptide comprises endoglucanase,phospholipase or chitosinase, preferably with at least 95% sequenceidentity to SEQ ID NO: 107, 108 and 109, respectively; and (iv) therecombinant Bacillus cereus family member cells comprise cells ofBacillus thuringiensis or Bacillus mycoides. In yet another particularembodiment, the recombinant Bacillus cereus family member cells arecells of Bacillus thuringiensis BT013A.

In a particular aspect of the above embodiments (i) the biologicalcontrol agent is Bacillus firmus I-1582 or mutants having all theidentifying characteristics of Bacillus firmus I-1582 and/or at least95% or at least 98% sequence identity to Bacillus firmus I-1582; (ii)the targeting sequence comprises an amino acid sequence having at leastabout 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 54%; (iii) the plantgrowth stimulating protein or peptide comprises endoglucanase,phospholipase or chitosinase, preferably with at least 95% or at least98% sequence identity to SEQ ID NO: 107, 108 and 109, respectively; and(iv) the recombinant Bacillus cereus family member cells comprise cellsof Bacillus thuringiensis or Bacillus mycoides. In yet anotherparticular embodiment, the recombinant Bacillus cereus family membercells are cells of Bacillus thuringiensis BT013A. In yet another aspectof this embodiment the composition further comprises clothianidin.

In a particular aspect of the above embodiments (i) the biologicalcontrol agent is Bacillus pumilus QST2808 or mutants having all theidentifying characteristics of Bacillus pumilus QST2808 and/or at least95% or at least 98% sequence identity to Bacillus pumilus QST2808; (ii)the targeting sequence comprises an amino acid sequence having at leastabout 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 54%; (iii) the plantgrowth stimulating protein or peptide comprises endoglucanase,phospholipase or chitosinase, preferably with at least 95% or at least98% sequence identity to SEQ ID NO: 107, 108 and 109, respectively; and(iv) the recombinant Bacillus cereus family member cells comprise thecells of Bacillus thuringiensis or Bacillus mycoides. In yet anotherparticular embodiment, the recombinant Bacillus cereus family membercells are cells of Bacillus thuringiensis BT013A.

In some aspects, the composition further comprises at least oneauxiliary selected from the group consisting of extenders, solvents,spontaneity promoters, carriers, emulsifiers, dispersants, frostprotectants, thickeners and adjuvants.

In other aspects, the invention is directed to a seed treated with anyof the compositions disclosed herein.

Furthermore, the present invention relates to use of the disclosedcompositions as a fungicide and/or insecticide. In certain aspects, thedisclosed compositions are used for reducing overall damage of plantsand plant parts as well as losses in harvested fruits or vegetablescaused by insects, mites, nematodes and/or phytopathogens. In otheraspects, the disclosed compositions are used for enhancing plant growthand/or promoting plant health.

Additionally, the present invention is directed to a method of treatinga plant, a plant part, such as a seed, root, rhizome, corm, bulb, ortuber, and/or a locus on which or near which the plant or the plantparts grow, such as soil, to enhance plant growth and/or promote planthealth comprising the step of simultaneously or sequentially applying toa plant, a plant part and/or a plant loci: a) recombinantexosporium-producing Bacillus cells that express a fusion proteincomprising: (i) at least one plant growth stimulating protein or peptideselected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound; anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource; and a protein or peptide that protects a plant from a pathogen;and (ii) a targeting sequence that localizes the fusion protein to theexosporium of the Bacillus cells; and b) at least one biological controlagent disclosed herein and/or a mutant of a specific strain of amicroorganism disclosed herein having all identifying characteristics ofthe respective strain, and/or at least one metabolite produced by therespective strain, and/or at least one metabolite produced by therespective strain that exhibits activity against insects, mites,nematodes and/or phytopathogens in a synergistically effective amount.

In another embodiment, the present invention is a method for reducingoverall damage of plants and plant parts as well as losses in harvestedfruits or vegetables caused by insects, mites, nematodes and/orphytopathogens comprising the step of simultaneously or sequentiallyapplying to a plant, a plant part, such as a seed, root, rhizome, corm,bulb, or tuber, and/or a locus on which or near which the plant or theplant parts grow, such as soil: a) recombinant exosporium-producingBacillus cells that express a fusion protein comprising: (i) at leastone plant growth stimulating protein or peptide selected from the groupconsisting of an enzyme involved in the production or activation of aplant growth stimulating compound; an enzyme that degrades or modifies abacterial, fungal, or plant nutrient source; and a protein or peptidethat protects a plant from a pathogen; and (ii) a targeting sequencethat localizes the fusion protein to the exosporium of the Bacilluscells; and b) at least one biological control agent disclosed hereinand/or a mutant of a specific strain of a microorganism disclosed hereinhaving all identifying characteristics of the respective strain, and/orat least one metabolite produced by the respective strain, and/or atleast one metabolite produced by the respective strain that exhibitsactivity against insects, mites, nematodes and/or phytopathogens in asynergistically effective amount.

In the above paragraphs, the term “comprise” or any derivative thereof(e.g., comprising, comprises) may be replaced with “consist of” or theapplicable corresponding derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequence of theamino-terminal portion of Bacillus anthracis Sterne strain Bc1A and withthe corresponding region from various exosporium proteins from Bacilluscereus family members.

DETAILED DESCRIPTION

In general “pesticidal” means the ability of a substance to increasemortality or inhibit the growth rate of plant pests. The term is usedherein, to describe the property of a substance to exhibit activityagainst insects, mites, nematodes and/or phytopathogens. In the sense ofthe present invention the term “pests” include insects, mites, nematodesand/or phytopathogens.

As used herein, “biological control” is defined as control of a pathogenand/or insect and/or an acarid and/or a nematode by the use of a secondorganism. Known mechanisms of biological control include bacteria thatcontrol root rot by out-competing fungi for space or nutrients on thesurface of the root. Bacterial toxins, such as antibiotics, have beenused to control pathogens. The toxin can be isolated and applieddirectly to the plant or the bacterial species may be administered so itproduces the toxin in situ. Other means of exerting biological controlinclude the application of certain fungi producing ingredients activeagainst a target phytopathogen, insect, mite or nematode, or attackingthe target pest/pathogen. “Biological control” as used in connectionwith the present invention may also encompass microorganisms having abeneficial effect on plant health, growth, vigor, stress response oryield. Application routes include spray application, soil applicationand seed treatment.

The term “metabolite” refers to any compound, substance or byproduct ofa fermentation of a said microorganism that has pesticidal, fungicidalor nematicidal activity or ability to enhance plant health or increaseplant yield. The term “mutant” refers to a variant of the parentalstrain as well as methods for obtaining a mutant or variant in which thepesticidal activity is greater than that expressed by the parentalstrain. The “parent strain” is defined herein as the original strainbefore mutagenesis or the deposited strain. To obtain such mutants theparental strain may be treated with a chemical such asN-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfone, or byirradiation using gamma, x-ray, or UV-irradiation, or by other meanswell known to those skilled in the art.

A “variant” is a strain having all the identifying characteristics ofthe NRRL or ATCC Accession Numbers as indicated in this text and can beidentified as having a genome that hybridizes under conditions of highstringency to the genome of the NRRL or ATCC Accession Numbers.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. Hybridization reactions can be performed underconditions of different “stringency”. In general, a low stringencyhybridization reaction is carried out at about 40° C. in 10×SSC or asolution of equivalent ionic strength/temperature. A moderate stringencyhybridization is typically performed at about 50° C. in 6×SSC, and ahigh stringency hybridization reaction is generally performed at about60° C. in 1×SSC.

A variant of the indicated NRRL or ATCC Accession Number may also bedefined as a strain having a genomic sequence that is greater than 85%,more preferably greater than 90% or more preferably greater than 95%sequence identity to the genome of the indicated NRRL or ATCC AccessionNumber. A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99%) of “sequence identity” to anothersequence means that, when aligned, that percentage of bases (or aminoacids) are the same in comparing the two sequences. This alignment andthe percent homology or sequence identity can be determined usingsoftware programs known in the art, for example, those described inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, Section 7. 7. 18, Table 7. 7. 1.

NRRL is the abbreviation for the Agricultural Research Service CultureCollection, having the address National Center for AgriculturalUtilization Research, Agricultural Research Service, U.S. Department ofAgriculture, 1815 North University Street, Peoria, Ill. 61604, U.S.A.

ATCC is the abbreviation for the American Type Culture Collection,having the address ATCC Patent Depository, 10801 University Boulevard,Manassas, Va. 10110, U.S.A.

CNCM is the abbreviation for the Collection Nationale de Cultures deMicroorganismes, Institute Pasteur, France, having the address ofInstitut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15,France.

All strains described herein and having an accession number in which theprefix is NRRL, ATCC or CNCM have been deposited with theabove-described respective depositary institution in accordance with theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

An “enzyme involved in the production or activation of a plant growthstimulating compound” includes any enzyme that catalyzes any step in abiological synthesis pathway for a compound that stimulates plant growthor alters plant structure, or any enzyme that catalyzes the conversionof an inactive or less active derivative of a compound that stimulatesplant growth or alters plant structure to an active or more active formof the compound. Such compounds include, for example, but are notlimited to, small molecule plant hormones such as auxins and cytokinins,bioactive peptides, and small plant growth stimulating moleculessynthesized by bacteria or fungi in the rhizosphere (e.g.,2,3-butanediol).

A “plant immune system enhancer protein or peptide” as used hereinincludes any protein or peptide that has a beneficial effect on theimmune system of a plant.

The term “plant growth stimulating protein or peptide” as used hereinincludes any protein or peptide that increases plant growth in a plantexposed to the protein or peptide.

The terms “promoting plant growth” and “stimulating plant growth” areused interchangeably herein, and refer to the ability to enhance orincrease at least one of the plant's height, weight, leaf size, rootsize, or stem size, to increase protein yield from the plant or toincrease grain yield of the plant.

A “protein or peptide that protects a plant from a pathogen” as usedherein includes any protein or peptide that makes a plant exposed to theprotein or peptide less susceptible to infection with a pathogen.

A “protein or peptide that enhances stress resistance in a plant” asused herein includes any protein or peptide that makes a plant exposedto the protein or peptide more resistant to stress.

The term “plant binding protein or peptide” refers to any peptide orprotein capable of specifically or non-specifically binding to any partof a plant (e.g., roots or aerial portions of a plant such as leavesfoliage, stems, flowers, or fruits) or to plant matter.

The term “targeting sequence” as used herein refers to a polypeptidesequence that results in the localization of a longer polypeptide or theprotein to the exosporium of a Bacillus cereus family member.

Recombinant Exosporium-Producing Bacillus Cells Expressing FusionProteins

The fusion proteins contain a targeting sequence, an exosporium protein,or an exosporium protein fragment that targets the fusion protein to theexosporium of a Bacillus cereus family member and: (a) a plant growthstimulating protein or peptide; (b) a protein or peptide that protects aplant from a pathogen; (c) a protein or peptide that enhances stressresistance of a plant; (d) a plant binding protein or peptide; or (e) aplant immune system enhancer protein or peptide. When expressed inBacillus cereus family member bacteria, these fusion proteins aretargeted to the exosporium layer of the spore and are physicallyoriented such that the protein or peptide is displayed on the outside ofthe spore.

This Bacillus exosporium display (BEMD) system can be used to deliverpeptides, enzymes, and other proteins to plants (e.g., to plant foliage,fruits, flowers, stems, or roots) or to a plant growth medium such assoil. Peptides, enzymes, and proteins delivered to the soil or anotherplant growth medium in this manner persist and exhibit activity in thesoil for extended periods of time. Introduction of recombinantexosporium-producing Bacillus cells expressing the fusion proteinsdescribed herein into soil or the rhizosphere of a plant leads to abeneficial enhancement of plant growth in many different soilconditions. The use of the BEMD to create these enzymes allows them tocontinue to exert their beneficial results to the plant and therhizosphere over the first months of a plants life.

Targeting Sequences, Exosporium Proteins, and Exosporium ProteinFragments

For ease of reference, the SEQ ID NOS. for the peptide and proteinsequences referred to herein are listed in Table 1 below.

TABLE 1 Peptide and Protein Sequences Sequence Identification Protein,Protein Fragment, or Targeting Sequence Number AA 1-41 of BclA SEQ IDNO: 1* (B. anthracis Sterne) Full length BclA SEQ ID NO: 2* AA 1-33 ofSEQ ID NO: 3 BetA/BAS3290 (B. anthracis Sterne) Full length BetA/BAS3290SEQ ID NO: 4 Met + AA 2-43 of SEQ ID NO: 5 BAS4623 (B. anthracis Sterne)Full length BAS4623 SEQ ID NO: 6 AA 1-34 of BclB SEQ ID NO: 7 (B.anthracis Sterne) Full length BclB SEQ ID NO: 8 AA 1-30 of BAS1882 (B.anthracis Sterne) SEQ ID NO: 9 Full length BAS1882 SEQ ID NO: 10 AA 1-39of gene 2280 (B. weihenstephensis KBAB4) SEQ ID NO: 11 Full length KBAB4gene 2280 SEQ ID NO: 12 AA 1-39 of gene 3572 (B. weihenstephensis KBAB4)SEQ ID NO: 13 Full Length KBAB4 gene 3572 SEQ ID NO: 14 AA 1-49 ofExosporium Leader Peptide SEQ ID NO: 15 (B. cereus VD200) Full LengthExosporium Leader Peptide SEQ ID NO: 16 AA 1-33 of Exosporium LeaderPeptide SEQ ID NO: 17 (B. cereus VD166) Full Length Exosporium LeaderPeptide SEQ ID NO: 18 AA 1-39 of hypothetical protein IKG_04663 SEQ IDNO: 19 (B. cereus VD200) Full Length hypothetical protein IKG_04663,partial SEQ ID NO: 20 AA 1-39 of YVTN β-propeller protein SEQ ID NO: 21(B. weihenstephensis KBAB4) Full length YVTN β-propeller protein KBAB4SEQ ID NO: 22 AA 1-30 of hypothetical protein bcerkbab4_2363 SEQ ID NO:23 (B. weihenstephensis KBAB4) Full length hypothetical proteinbcerkbab4_2363 SEQ ID NO: 24 KBAB4 AA 1-30 of hypothetical proteinbcerkbab4_2131 SEQ ID NO: 25 (B. weihenstephensis KBAB4) Full lengthhypothetical protein bcerkbab4_2131 SEQ ID NO: 26 AA 1-36 of triplehelix repeat containing collagen SEQ ID NO: 27 (B. weihenstephensisKBAB4) Full length triple helix repeat-containing SEQ ID NO: 28 collagenKBAB4 AA 1-39 of hypothetical protein bmyco0001_21660 SEQ ID NO: 29 (B.mycoides 2048) Full length hypothetical protein bmyco0001_21660 SEQ IDNO: 30 AA 1-30 of hypothetical protein bmyc0001_22540 SEQ ID NO: 31 (B.mycoides 2048) Full length hypothetical protein bmyc0001_22540 SEQ IDNO: 32 AA 1-21 of hypothetical protein bmyc0001_21510 SEQ ID NO: 33 (B.mycoides 2048) Full length hypothetical protein bmyc0001_21510 SEQ IDNO: 34 AA 1-22 of collagen triple helix repeat protein SEQ ID NO: 35 (B.thuringiensis 35646) Full length collagen triple helix repeat proteinSEQ ID NO: 36 AA 1-35 of hypothetical protein WP_69652 SEQ ID NO: 43 (B.cereus) Full length hypothetical protein WP_69652 SEQ ID NO: 44 AA 1-41of exosporium leader WP016117717 SEQ ID NO: 45 (B. cereus) Full lengthexosporium leader WP016117717 SEQ ID NO: 46 AA 1-49 of exosporiumpeptide WP002105192 SEQ ID NO: 47 (B. cereus) Full length exosporiumpeptide WP002105192 SEQ ID NO: 48 AA 1-38 of hypothetical proteinWP87353 SEQ ID NO: 49 (B. cereus) Full length hypothetical proteinWP87353 SEQ ID NO: 50 AA 1-39 of exosporium peptide 02112369 SEQ ID NO:51 (B. cereus) Full length exosporium peptide 02112369 SEQ ID NO: 52 AA1-39 of exosporium protein WP016099770 SEQ ID NO: 53 (B. cereus) Fulllength exosporium protein WP016099770 SEQ ID NO: 54 AA 1-36 ofhypothetical protein YP006612525 SEQ ID NO: 55 (B. thuringiensis) Fulllength hypothetical protein YP006612525 SEQ ID NO: 56 AA 1-136 ofhypothetical protein TIGR03720 SEQ ID (B. mycoides) NO: 57** Full lengthhypothetical protein TIGR03720 SEQ ID NO: 58** AA 1-196 of BclA SEQ IDNO: 59* (B. anthracis Sterne) Met + AA 20-35 of BclA SEQ ID NO: 60 (B.anthracis Sterne) Met + AA 12-27 of BetA/BAS3290 SEQ ID NO: 61 (B.anthracis Sterne) Met + AA 18-33 of gene 2280 SEQ ID NO: 62 (B.weihenstephensis KBAB4) Met + AA 18-33 of gene 3572 SEQ ID NO: 63 (B.weihenstephensis KBAB4) Met + AA 12-27 of Exosporium Leader Peptide SEQID NO: 64 (B. cereus VD166) Met + AA 18-33 of YVTN β-propeller proteinSEQ ID NO: 65 (B. weihenstephensis KBAB4) Met + AA 9-24 of hypotheticalprotein SEQ ID NO: 66 bcerkbab4_2363 (B. weihenstephensis KBAB4) Met +AA 9-24 of hypothetical protein SEQ ID NO: 67 bcerkbab4_2131 (B.weihenstephensis KBAB4) Met + AA 9-24 of hypothetical protein SEQ ID NO:68 bmyc0001_22540 (B. mycoides 2048) Met + AA 9-24 of SEQ ID NO: 69BAS1882 (B. anthracis Sterne) Met + AA 20-35 of exosporium leaderWP016117717 SEQ ID NO: 70 (B. cereus) Full length InhA SEQ ID NO: 71 (B.mycoides) Full length BAS1141 (ExsY) SEQ ID NO: 72 (B. anthracis Sterne)Full length BAS1144 (BxpB/ExsFA) SEQ ID NO: 73 (B. anthracis Sterne)Full length BAS1145 (CotY) SEQ ID NO: 74 (B. anthracis Sterne) Fulllength BAS1140 SEQ ID NO: 75 (B. anthracis Sterne) Full length ExsFB SEQID NO: 76 (B. anthracis H9401) Full length InhA1 SEQ ID NO: 77 (B.thuringiensis HD74) Full length ExsJ SEQ ID NO: 78 (B. cereus ATCC10876) Full length ExsH SEQ ID NO: 79 (B. cereus) Full length YjcA SEQID NO: 80 (B. anthracis Ames) Full length YjcB SEQ ID NO: 81 (B.anthracis) Full length BclC SEQ ID NO: 82 (B. anthracis Sterne) Fulllength acid phosphatase SEQ ID NO: 83 (Bacillus thuringiensis serovarkonkukian str. 97-27) Full length InhA2 SEQ ID NO: 84 (B. thuringiensisHD74) AA = amino acids *B. anthracis Sterne strain BclA has 100%sequence identity with B. thuringiensis BclA. Thus, SEQ ID NOS: 1, 2,and 59 also represent amino acids 1-41 of B. thuringiensis BclA, fulllength B. thuringiensis BclA, and amino acids 1-196 of B. thuringiensisBclA, respectively. Likewise, SEQ ID NO: 60 also represents a methionineresidue plus amino acids 20-35 of B. thuringiensis BclA. **B. mycoideshypothetical protein TIGR03720 has 100% sequence identity with B.mycoides hypothetical protein WP003189234. Thus, SEQ ID NOs: 57 and 58also represent amino acids 1-136 of B. mycoides hypothetical proteinWP003189234 and full length B. mycoides hypothetical proteinWP003189234, respectively.

Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus familyof bacteria includes the species Bacillus anthracis, Bacillus cereus,Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides,Bacillus samanii, Bacillus gaemokensis, Bacillus toyoiensis and Bacillusweihenstephensis. Under stressful environmental conditions, Bacilluscereus family bacteria undergo sporulation and form oval endospores thatcan stay dormant for extended periods of time. The outermost layer ofthe endospores is known as the exosporium and comprises a basal layersurrounded by an external nap of hair-like projections. Filaments on thehair-like nap are predominantly formed by the collagen-like glycoproteinBc1A, while the basal layer is comprised of a number of differentproteins. Another collagen-related protein, Bc1B, is also present in theexosporium and exposed on endospores of Bacillus cereus family members.

Bc1A, the major constituent of the surface nap, has been shown to beattached to the exosporium with its amino-terminus (N-terminus)positioned at the basal layer and its carboxy-terminus (C-terminus)extending outward from the spore.

It was previously discovered that certain sequences from the N-terminalregions of Bc1A and Bc1B could be used to target a peptide or protein tothe exosporium of a Bacillus cereus endospore (see U.S. PatentPublication Nos. 2010/0233124 and 2011/0281316, and Thompson, et al.,“Targeting of the Bc1A and Bc1B Proteins to the Bacillus anthracis SporeSurface,” Molecular Microbiology, 70(2):421-34 (2008), the entirety ofeach of which is hereby incorporated by reference). It was also foundthat the BetA/BAS3290 protein of Bacillus anthracis localized to theexosporium.

In particular, amino acids 20-35 of Bc1A from Bacillus anthracis Sternestrain have been found to be sufficient for targeting to the exosporium.A sequence alignment of amino acids 1-41 of Bc1A (SEQ ID NO: 1) with thecorresponding N-terminal regions of several other Bacillus cereus familyexosporium proteins and Bacillus cereus family proteins having relatedsequences is shown in FIG. 1. As can be seen from FIG. 1, there is aregion of high-homology among all of the proteins in the regioncorresponding to amino acids 20-41 of Bc1A. However, in these sequences,the amino acids corresponding to amino acids 36-41 of Bc1A containsecondary structure and are not necessary for fusion proteinlocalization to the exosporium. The conserved targeting sequence regionof Bc1A (amino acids 20-35 of SEQ ID NO: 1) is shown in bold in FIG. 1and corresponds to the minimal targeting sequence needed forlocalization to the exosporium. A more highly conserved region spanningamino acids 25-35 of Bc1A within the targeting sequence is underlined inthe sequences in FIG. 1, and is the recognition sequence forExsFA/BxpB/ExsFB and homologs, which direct and assemble the describedproteins on the surface of the exosporium The amino acid sequences ofSEQ ID NOS: 3, 5, and 7 in FIG. 1 are amino acids 1-33 of Bacillusanthracis Sterne strain BetA/BAS3290, a methionine followed by aminoacids 2-43 of Bacillus anthracis Sterne strain BAS4623, and amino acids1-34 of Bacillus anthracis Sterne strain Bc1B, respectively. (ForBAS4623, it was found that replacing the valine present at position 1 inthe native protein with a methionine resulted in better expression.) Ascan be seen from FIG. 1, each of these sequences contains a conservedregion corresponding to amino acids 20-35 of Bc1A (SEQ ID NO: 1; shownin bold), and a more highly conserved region corresponding to aminoacids 20-35 of Bc1A (underlined).

Additional proteins from Bacillus cereus family members also contain theconserved targeting region. In particular, in FIG. 1, SEQ ID NO: 9 isamino acids 1-30 of Bacillus anthracis Sterne strain BAS1882, SEQ ID NO:11 is amino acids 1-39 of the Bacillus weihenstephensis KBAB4 2280 geneproduct, SEQ ID NO: 13 is amino acids 1-39 of the Bacillusweihenstephensis KBAB4 3572 gene product, SEQ ID NO: 15 is amino acids1-49 of Bacillus cereus VD200 exosporium leader peptide, SEQ ID NO: 17is amino acids 1-33 of Bacillus cereus VD166 exosporium leader peptide,SEQ ID NO: 19 is amino acids 1-39 of Bacillus cereus VD200 hypotheticalprotein IKG_04663, SEQ ID NO: 21 is amino acids 1-39 of Bacillusweihenstephensis KBAB4 YVTN β-propeller protein, SEQ ID NO: 23 is aminoacids 1-30 of Bacillus weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, SEQ ID NO: 25 is amino acids 1-30 of Bacillusweihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, SEQ ID NO:27 is amino acids 1-36 of Bacillus weihenstephensis KBAB4 triple helixrepeat containing collagen, SEQ ID NO: 29 is amino acids 1-39 ofBacillus mycoides 2048 hypothetical protein bmyco0001_21660, SEQ ID NO:31 is amino acids 1-30 of Bacillus mycoides 2048 hypothetical proteinbmyc0001_22540, SEQ ID NO: 33 is amino acids 1-21 of Bacillus mycoides2048 hypothetical protein bmyc0001_21510, SEQ ID NO: 35 is amino acids1-22 of Bacillus thuringiensis 35646 collagen triple helix repeatprotein, SEQ ID NO: 43 is amino acids 1-35 of Bacillus cereushypothetical protein WP_69652, SEQ ID NO: 45 is amino acids 1-41 ofBacillus cereus exosporium leader WP016117717, SEQ ID NO: 47 is aminoacids 1-49 of Bacillus cereus exosporium peptide WP002105192, SEQ ID NO:49 is amino acids 1-38 of Bacillus cereus hypothetical protein WP87353,SEQ ID NO: 51 is amino acids 1-39 of Bacillus cereus exosporium peptide02112369, SEQ ID NO: 53 is amino acids 1-39 of Bacillus cereusexosporium protein WP016099770, SEQ ID NO: 55 is amino acids 1-36 ofBacillus thuringiensis hypothetical protein YP006612525, and SEQ ID NO:57 is amino acids 1-136 of Bacillus mycoides hypothetical proteinTIGR03720. As shown in FIG. 1, each of the N-terminal regions of theseproteins contains a region that is conserved with amino acids 20-35 ofBc1A (SEQ ID NO: 1), and a more highly conserved region corresponding toamino acids 25-35 of Bc1A.

Any portion of Bc1A which includes amino acids 20-35 can be used as thetargeting sequence. In addition, full-length exosporium proteins orexosporium protein fragments can be used for targeting the fusionproteins to the exosporium. Thus, full-length Bc1A or a fragment of Bc1Athat includes amino acids 20-35 can be used for targeting to theexosporium. For example, full length Bc1A (SEQ ID NO: 2) or a midsizedfragment of Bc1A that lacks the carboxy-terminus such as SEQ ID NO: 59(amino acids 1-196 of Bc1A) can be used to target the fusion proteins tothe exosporium. Midsized fragments such as the fragment of SEQ ID NO: 59have less secondary structure than full length Bc1A and have been foundto be suitable for use as a targeting sequence. The targeting sequencecan also comprise much shorter portions of Bc1A which include aminoacids 20-35, such as SEQ ID NO: 1 (amino acids 1-41 of Bc1A), aminoacids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1, or SEQ IDNO: 60 (a methionine residue linked to amino acids 20-35 of Bc1A). Evenshorter fragments of Bc1A which include only some of amino acids 20-35also exhibit the ability to target fusion proteins to the exosporium.For example, the targeting sequence can comprise amino acids 22-31 ofSEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or amino acids 20-31 ofSEQ ID NO: 1.

Alternatively, any portion of BetA/BAS3290, BAS4623, Bc1B, BAS1882, theKBAB4 2280 gene product, the KBAB4 3572 gene product, B. cereus VD200exosporium leader peptide, B. cereus VD166 exosporium leader peptide, B.cereus VD200 hypothetical protein IKG_04663, B. weihenstephensis KBAB4YVTN β-propeller protein, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131, B. weihenstephensis KBAB4 triple helix repeat containingcollagen, B. mycoides 2048 hypothetical protein bmyco0001_21660, B.mycoides 2048 hypothetical protein bmyc0001_22540, B. mycoides 2048hypothetical protein bmyc0001_21510, B. thuringiensis 35646 collagentriple helix repeat protein, B. cereus hypothetical protein WP_69652, B.cereus exosporium leader WP016117717, B. cereus exosporium peptideWP002105192, B. cereus hypothetical protein WP87353, B. cereusexosporium peptide 02112369, B. cereus exosporium protein WP016099770,B. thuringiensis hypothetical protein YP006612525, or B. mycoideshypothetical protein TIGR03720, which includes the amino acidscorresponding to amino acids 20-35 of Bc1A can serve as the targetingsequence. As can be seen from FIG. 1, amino acids 12-27 of BetA/BAS3290,amino acids 23-38 of BAS4623, amino acids 13-28 of Bc1B, amino acids9-24 of BAS1882, amino acids 18-33 of KBAB4 2280 gene product, aminoacids 18-33 of KBAB4 3572 gene product, amino acids 28-43 of B. cereusVD200 exosporium leader peptide, amino acids 12-27 of B. cereus VD166exosporium leader peptide, amino acids 18-33 of B. cereus VD200hypothetical protein IKG_04663, amino acids 18-33 B. weihenstephensisKBAB4 YVTN β-propeller protein, amino acids 9-24 of B. weihenstephensisKBAB4 hypothetical protein bcerkbab4_2363, amino acids 9-24 of B.weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, amino acids15-30 of B. weihenstephensis KBAB4 triple helix repeat containingcollagen, amino acids 18-33 of B. mycoides 2048 hypothetical proteinbmyco0001_21660, amino acids 9-24 of B. mycoides 2048 hypotheticalprotein bmyc0001_22540, amino acids 1-15 of B. mycoides 2048hypothetical protein bmyc0001_21510, amino acids 1-16 of B.thuringiensis 35646 collagen triple helix repeat protein, amino acids14-29 of B. cereus hypothetical protein WP_69652, amino acids 20-35 ofB. cereus exosporium leader WP016117717, amino acids 28-43 of B. cereusexosporium peptide WP002105192, amino acids 17-32 of B. cereushypothetical protein WP87353, amino acids 18-33 of B. cereus exosporiumpeptide 02112369, amino acids 18-33 of B. cereus exosporium proteinWP016099770, amino acids 15-30 of B. thuringiensis hypothetical proteinYP006612525, and amino acids 115-130 of B. mycoides hypothetical proteinTIGR03720 correspond to amino acids 20-35 of Bc1A. Thus, any portion ofthese proteins that includes the above-listed corresponding amino acidscan serve as the targeting sequence.

Furthermore, any amino acid sequence comprising amino acids 20-35 ofBc1A, or any of the above-listed corresponding amino acids can serve asthe targeting sequence.

Thus, the targeting sequence can comprise amino acids 1-35 of SEQ ID NO:1, amino acids 20-35 of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 60, aminoacids 22-31 of SEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or aminoacids 20-31 of SEQ ID NO: 1. Alternatively, the targeting sequenceconsists of amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 60. Alternatively, the targetingsequence can consist of amino acids 22-31 of SEQ ID NO: 1, amino acids22-33 of SEQ ID NO: 1, or amino acids 20-31 of SEQ ID NO: 1.Alternatively, the exosporium protein can comprise full length Bc1A (SEQID NO: 2), or the exosporium protein fragment can comprise a midsizedfragment of Bc1A that lacks the carboxy-terminus, such as SEQ ID NO: 59(amino acids 1-196 of Bc1A). Alternatively, the exosporium proteinfragment can consist of SEQ ID NO: 59.

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:3, amino acids 12-27 of SEQ ID NO: 3, or SEQ ID NO: 3, or the exosporiumprotein can comprise full length BetA/BAS3290 (SEQ ID NO: 4). It hasalso been found that a methionine residue linked to amino acids 12-27 ofBetA/BAS3290 can be used as a targeting sequence. Thus, the targetingsequence can comprise SEQ ID NO: 61. The targeting sequence can alsocomprise amino acids 14-23 of SEQ ID NO: 3, amino acids 14-25 of SEQ IDNO: 3, or amino acids 12-23 of SEQ ID NO: 3.

The targeting sequence can also comprise amino acids 1-38 of SEQ ID NO:5, amino acids 23-38 of SEQ ID NO: 5, or SEQ ID NO: 5, or the exosporiumprotein can comprise full length BAS4623 (SEQ ID NO: 6).

Alternatively, the targeting sequence can comprise amino acids 1-28 ofSEQ ID NO: 7, amino acids 13-28 of SEQ ID NO: 7, or SEQ ID NO: 7, or theexosporium protein can comprise full length Bc1B (SEQ ID NO: 8).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:9, amino acids 9-24 of SEQ ID NO: 9, or SEQ ID NO: 9, or the exosporiumprotein can comprise full length BAS1882 (SEQ ID NO: 10). A methionineresidue linked to amino acids 9-24 of BAS1882 can also be used as atargeting sequence. Thus, the targeting sequence can comprise SEQ ID NO:69.

The targeting sequence can also comprise amino acids 1-33 of SEQ IDNO:11, amino acids 18-33 of SEQ ID NO: 11, or SEQ ID NO: 11, or theexosporium protein can comprise the full length B. weihenstephensisKBAB4 2280 gene product (SEQ ID NO: 12). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 2280 gene product canalso be used as a targeting sequence. Thus, the targeting sequence cancomprise SEQ ID NO: 62.

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:13, amino acids 18-33 of SEQ ID NO: 13, or SEQ ID NO:13, or theexosporium protein can comprise the full length B. weihenstephensisKBAB4 3572 gene product (SEQ ID NO:14). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 3572 gene product canalso be used as a targeting sequence. Thus, the targeting sequence cancomprise SEQ ID NO: 63.

Alternatively, the targeting sequence can comprise amino acids 1-43 ofSEQ ID NO: 15, amino acids 28-43 of SEQ ID NO: 15, or SEQ ID NO: 15, orthe exosporium protein can comprise full length B. cereus VD200exosporium leader peptide (SEQ ID NO: 16).

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:17, amino acids 12-27 of SEQ ID NO: 17, or SEQ ID NO: 17, or theexosporium protein can comprise full-length B. cereus VD166 exosporiumleader peptide (SEQ ID NO: 18). A methionine residue linked to aminoacids 12-27 of the B. cereus VD166 exosporium leader peptide can also beused as a targeting sequence. Thus, the targeting sequence can compriseSEQ ID NO: 64.

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:19, amino acids 18-33 of SEQ ID NO: 19, or SEQ ID NO: 19, or theexosporium protein can comprise full length B. cereus VD200 hypotheticalprotein IKG_04663 (SEQ ID NO: 20).

Alternatively, the targeting sequence comprises amino acids 1-33 of SEQID NO: 21, amino acids 18-33 of SEQ ID NO: 21, or SEQ ID NO: 21, or theexosporium protein can comprise full length B. weihenstephensis KBAB4YVTN β-propeller protein (SEQ ID NO: 22). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 YVTN β-propellerprotein can also be used as a targeting sequence. Thus, the targetingsequence can comprise SEQ ID NO: 65.

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:23, amino acids 9-24 of SEQ ID NO: 23, or SEQ ID NO: 23, or theexosporium protein can comprise full length B. weihenstephensis KBAB4hypothetical protein bcerkbab4_2363 (SEQ ID NO: 24). A methionineresidue linked to amino acids 9-24 of B. weihenstephensis KBAB4hypothetical protein bcerkbab4_2363 can also be used as a targetingsequence. Thus, the targeting sequence can comprise SEQ ID NO: 66.

The targeting sequence comprise amino acids 1-24 of SEQ ID NO: 25, aminoacids 9-24 of SEQ ID NO: 25, or SEQ ID NO: 25, or the exosporium proteincan comprise full length B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131 (SEQ ID NO: 26). A methionine residue linked to aminoacids 9-24 of B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131 can also be used as a targeting sequence. Thus, thetargeting sequence can comprise SEQ ID NO: 67.

Alternatively, the targeting sequence comprises amino acids 1-30 of SEQID NO: 27, amino acids 15-30 of SEQ ID NO: 27, or SEQ ID NO: 27, or theexosporium protein can comprise full length B. weihenstephensis KBAB4triple helix repeat containing collagen (SEQ ID NO: 28).

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:29, amino acids 18-33 of SEQ ID NO: 29, or SEQ ID NO: 29, or theexosporium protein can comprise full length B. mycoides 2048hypothetical protein bmyco0001_21660 (SEQ ID NO:30).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:31, amino acids 9-24 of SEQ ID NO: 31, or SEQ ID NO:31, or theexosporium protein can comprise full length B. mycoides 2048hypothetical protein bmyc0001_22540 (SEQ ID NO:32). A methionine residuelinked to amino acids 9-24 of B. mycoides 2048 hypothetical proteinbmyc0001_22540 can also be used as a targeting sequence. Thus, thetargeting sequence can comprise SEQ ID NO: 68.

Alternatively, the targeting sequence comprises amino acids 1-15 of SEQID NO: 33, SEQ ID NO: 33, or the exosporium protein comprises fulllength B. mycoides 2048 hypothetical protein bmyc0001_21510 (SEQ ID NO:34).

The targeting sequence can also comprise amino acids 1-16 of SEQ ID NO:35, SEQ ID NO: 35, or the exosporium protein can comprise full length B.thuringiensis 35646 collagen triple helix repeat protein (SEQ ID NO:36).

The targeting sequence can comprise amino acids 1-29 of SEQ ID NO: 43,amino acids 14-29 of SEQ ID NO: 43, or SEQ ID NO: 43, or the exosporiumprotein can comprise full length B. cereus hypothetical protein WP_69652(SEQ ID NO: 44).

Alternatively, the targeting sequence can comprise amino acids 1-35 ofSEQ ID NO: 45, amino acids 20-35 of SEQ ID NO: 45, or SEQ ID NO: 45, orthe exosporium protein can comprise full length B. cereus exosporiumleader WP016117717 (SEQ ID NO: 46). A methionine residue linked to aminoacids 20-35 of B. cereus exosporium leader WP016117717 can also be usedas a targeting sequence. Thus, the targeting sequence can comprise SEQID NO: 70.

The targeting sequence can comprise amino acids 1-43 of SEQ ID NO: 47,amino acids 28-43 of SEQ ID NO: 47, or SEQ ID NO: 47, or the exosporiumprotein can comprise full length B. cereus exosporium peptideWP002105192 (SEQ ID NO: 48).

The targeting sequence can comprise amino acids 1-32 of SEQ ID NO: 49,amino acids 17-32 of SEQ ID NO: 49, or SEQ ID NO: 49, or the exosporiumprotein can comprise full length B. cereus hypothetical protein WP87353(SEQ ID NO: 50).

Alternatively, the targeting sequence can comprise amino acids 1-33 ofSEQ ID NO: 51, amino acids 18-33 of SEQ ID NO: 51, or SEQ ID NO: 51, orthe exosporium protein can comprise full length B. cereus exosporiumpeptide 02112369 (SEQ ID NO: 52).

The targeting sequence can comprise amino acids 1-33 of SEQ ID NO: 53,amino acids 18-33 of SEQ ID NO: 53, or SEQ ID NO: 53, or the exosporiumprotein can comprise full length B. cereus exosporium proteinWP016099770 (SEQ ID NO: 54).

Alternatively, the targeting sequence can comprise acids 1-30 of SEQ IDNO: 55, amino acids 15-30 of SEQ ID NO: 55, or SEQ ID NO: 55, or theexosporium protein can comprise full length B. thuringiensishypothetical protein YP006612525 (SEQ ID NO: 56).

The targeting sequence can also comprise amino acids 1-130 of SEQ ID NO:57, amino acids 115-130 of SEQ ID NO: 57, or SEQ ID NO: 57, or theexosporium protein can comprise full length B. mycoides hypotheticalprotein TIGR03720 (SEQ ID NO: 58).

In addition, it can readily be seen from the sequence alignment in FIG.1 that while amino acids 20-35 of Bc1A are conserved, and amino acids25-35 are more conserved, some degree of variation can occur in thisregion without affecting the ability of the targeting sequence to targeta protein to the exosporium. FIG. 1 lists the percent identity of eachof corresponding amino acids of each sequence to amino acids 20-35 ofBc1A (“20-35% Identity”) and to amino acids 25-35 of Bc1A (“25-35%Identity”). Thus, for example, as compared to amino acids 20-35 of Bc1A,the corresponding amino acids of BetA/BAS3290 are about 81.3% identical,the corresponding amino acids of BAS4623 are about 50.0% identical, thecorresponding amino acids of Bc1B are about 43.8% identical, thecorresponding amino acids of BAS1882 are about 62.5% identical, thecorresponding amino acids of the KBAB4 2280 gene product are about 81.3%identical, and the corresponding amino acids of the KBAB4 3572 geneproduct are about 81.3% identical. The sequence identities over thisregion for the remaining sequences are listed in FIG. 1.

With respect to amino acids 25-35 of Bc1A, the corresponding amino acidsof BetA/BAS3290 are about 90.9% identical, the corresponding amino acidsof BAS4623 are about 72.7% identical, the corresponding amino acids ofBc1B are about 54.5% identical, the corresponding amino acids of BAS1882are about 72.7% identical, the corresponding amino acids of the KBAB42280 gene product are about 90.9% identical, and the corresponding aminoacids of the KBAB4 3572 gene product are about 81.8% identical. Thesequence identities over this region for the remaining sequences arelisted in FIG. 1.

Thus, the targeting sequence can comprise an amino acid sequence havingat least about 43% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 54%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 43% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 54%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 72%.

The targeting sequence can also comprise an amino acid sequence havingat least about 56% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 56% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

Alternatively, the targeting sequence can comprise an amino sequencehaving at least about 62% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 72%.The targeting sequence can also consist of an amino acid sequenceconsisting of 16 amino acids and having at least about 62% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 72%.

The targeting sequence can comprise an amino acid sequence having atleast 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 81%. Alternatively,the targeting sequence consists of an amino acid sequence consisting of16 amino acids and having at least 68% identity with amino acids 20-35of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at leastabout 81%.

The targeting sequence can also comprise an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 75% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 72%.

The targeting sequence can also comprise an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 75% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 81%.

The targeting sequence can also comprise an amino acid sequence havingat least about 81% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 81%.

The targeting sequence can comprise an amino acid sequence having atleast about 81% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 90%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 90%.

The skilled person will recognize that variants of the above sequencescan also be used as targeting sequences, so long as the targetingsequence comprises amino acids 20-35 of Bc1A, the corresponding aminoacids of BetA/BAS3290, BAS4263, Bc1B, BAS1882, the KBAB4 2280 geneproduct, or the KBAB 3572 gene product, or a sequence comprising any ofthe above noted sequence identities to amino acids 20-35 and 25-35 ofBc1A is present.

It has further been discovered that certain Bacillus cereus familyexosporium proteins which lack regions having homology to amino acids25-35 of Bc1A can also be used to target a peptide or protein to theexosporium of a Bacillus cereus family member. In particular, the fusionproteins can comprise an exosporium protein comprising SEQ ID NO: 71 (B.mycoides InhA), an exosporium protein comprising SEQ ID NO: 72 (B.anthracis Sterne BAS1141 (ExsY)), an exosporium protein comprising SEQID NO: 73 (B. anthracis Sterne BAS1144 (BxpB/ExsFA)), an exosporiumprotein comprising SEQ ID NO: 74 (B. anthracis Sterne BAS1145 (CotY)),an exosporium protein comprising SEQ ID NO: 75 (B. anthracis SterneBAS1140), an exosporium protein comprising SEQ ID NO: 76 (B. anthracisH9401 ExsFB), an exosporium protein comprising SEQ ID NO: 77 (B.thuringiensis HD74 InhA1), an exosporium protein comprising SEQ ID NO:78 (B. cereus ATCC 10876 ExsJ), an exosporium protein comprising SEQ IDNO: 79 (B. cereus ExsH), an exosporium protein comprising SEQ ID NO: 80(B. anthracis Ames YjcA), an exosporium protein comprising SEQ ID NO: 81(B. anthracis YjcB), an exosporium protein comprising SEQ ID NO: 82 (B.anthracis Sterne Bc1C), an exosporium protein comprising SEQ ID NO: 83(Bacillus thuringiensis serovar konkukian str. 97-27 acid phosphatase),or an exosporium protein comprising SEQ ID NO: 84 (B. thuringiensis HD74InhA2). Inclusion of an exosporium protein comprising SEQ ID NO: 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 in the fusion proteinsdescribed herein will result in targeting to the exosporium of a B.cereus family member.

Moreover, exosporium proteins having a high degree of sequence identitywith any of the full-length exosporium proteins or the exosporiumprotein fragments described above can also be used to target a peptideor protein to the exosporium of a Bacillus cereus family member. Thus,the fusion protein can comprise an exosporium protein comprising anamino acid sequence having at least 85% identity with any one of SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,44, 46, 48, 50, 52, 54, 56, 58, 59, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, and 84. Alternatively, the fusion protein can comprisean exosporium protein having at least 90%, at least 95%, at least 98%,at least 99%, or 100% identity with any one of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50,52, 54, 56, 58, 59, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,and 84.

Alternatively, the fusion protein can comprise an exosporium proteinfragment consisting of an amino acid sequence having at least 85%identity with SEQ ID NO: 59. Alternatively, the fusion protein cancomprise an exosporium protein fragment consisting of an amino acidsequence having at least 90%, at least 95%, at least 98%, at least 99%,or 100% identity with SEQ ID NO: 59.

In any of the targeting sequences, exosporium proteins, or exosporiumprotein fragments described herein, the targeting sequence, exosporiumprotein, or exosporium protein fragment can comprise the amino acidsequence GXT at its carboxy terminus, wherein X is any amino acid.

In any of the targeting sequences, exosporium proteins, and exosporiumprotein fragments described herein, the targeting sequence, exosporiumprotein, or exosporium protein fragment, can comprise an alanine residueat the position of the targeting sequence that corresponds to amino acid20 of SEQ ID NO: 1.

Fusion Proteins

The fusion proteins can comprise a targeting sequence, an exosporiumprotein, or an exosporium protein fragment, and at least one plantgrowth stimulating protein or peptide. The plant growth stimulatingprotein or peptide can comprise a peptide hormone, a non-hormonepeptide, an enzyme involved in the production or activation of a plantgrowth stimulating compound or an enzyme that degrades or modifies abacterial, fungal, or plant nutrient source. The targeting sequence,exosporium protein, or exosporium protein fragment can be any of thetargeting sequences, exosporium proteins, or exosporium proteinfragments described above.

The fusion proteins can comprise a targeting sequence, an exosporiumprotein, or an exosporium protein fragment, and at least one protein orpeptide that protects a plant from a pathogen. The targeting sequence,exosporium protein, or exosporium protein fragment can be any of thetargeting sequences, exosporium proteins, or exosporium proteinfragments described above.

The fusion protein can be made using standard cloning and molecularbiology methods known in the art. For example, a gene encoding a proteinor peptide (e.g., a gene encoding a plant growth stimulating protein orpeptide) can be amplified by polymerase chain reaction (PCR) and ligatedto DNA coding for any of the above-described targeting sequences to forma DNA molecule that encodes the fusion protein. The DNA moleculeencoding the fusion protein can be cloned into any suitable vector, forexample a plasmid vector. The vector suitably comprises a multiplecloning site into which the DNA molecule encoding the fusion protein canbe easily inserted. The vector also suitably contains a selectablemarker, such as an antibiotic resistance gene, such that bacteriatransformed, transfected, or mated with the vector can be readilyidentified and isolated. Where the vector is a plasmid, the plasmidsuitably also comprises an origin of replication. The DNA encoding thefusion protein is suitably under the control of a sporulation promoterwhich will cause expression of the fusion protein on the exosporium of aB. cereus family member endospore (e.g., a native bc1A promoter from aB. cereus family member). Alternatively, DNA coding for the fusionprotein can be integrated into the chromosomal DNA of the B. cereusfamily member host.

The fusion protein can also comprise additional polypeptide sequencesthat are not part of the targeting sequence, exosporium protein,exosporium protein fragment, or the plant growth stimulating protein orpeptide, the protein or peptide that protects a plant from a pathogen,the protein or peptide that enhances stress resistance in a plant, orthe plant binding protein or peptide. For example, the fusion proteincan include tags or markers to facilitate purification or visualizationof the fusion protein (e.g., a polyhistidine tag or a fluorescentprotein such as GFP or YFP) or visualization of recombinantexosporium-producing Bacillus cells spores expressing the fusionprotein.

Expression of fusion proteins on the exosporium using the targetingsequences, exosporium proteins, and exosporium protein fragmentsdescribed herein is enhanced due to a lack of secondary structure in theamino-termini of these sequences, which allows for native folding of thefused proteins and retention of activity. Proper folding can be furtherenhanced by the inclusion of a short amino acid linker between thetargeting sequence, exosporium protein, exosporium protein fragment, andthe fusion partner protein.

Thus, any of the fusion proteins described herein can comprise an aminoacid linker between the targeting sequence, the exosporium protein, orthe exosporium protein fragment and the plant growth stimulating proteinor peptide, the protein or peptide that protects a plant from apathogen, the protein or peptide that enhances stress resistance in aplant, or the plant binding protein or peptide.

The linker can comprise a polyalanine linker or a polyglycine linker. Alinker comprising a mixture of both alanine and glycine residues canalso be used. For example, where the targeting sequence comprises SEQ IDNO: 1, a fusion protein can have one of the following structures:

No linker: SEQ ID NO: 1—Fusion Partner Protein

Alanine Linker: SEQ ID NO: 1—An-Fusion Partner Protein

Glycine Linker: SEQ ID NO: 1—Gn-Fusion Partner Protein

Mixed Alanine and Glycine Linker: SEQ ID NO: 1—(A/G)n-Fusion PartnerProtein

where An, Gn, and (A/G)n are any number of alanines, any number ofglycines, or any number of a mixture of alanines and glycines,respectively. For example, n can be 1 to 25, and is preferably 6 to 10.Where the linker comprises a mixture of alanine and glycine residues,any combination of glycine and alanine residues can be used. In theabove structures, “Fusion Partner Protein” represents the plant growthstimulating protein or peptide, the protein or peptide that protects aplant from a pathogen, the protein or peptide that enhances stressresistance in a plant, or the plant binding protein or peptide.

Alternatively or in addition, the linker can comprise a proteaserecognition site. Inclusion of a protease recognition site allows fortargeted removal, upon exposure to a protease that recognizes theprotease recognition site, of the plant growth stimulating protein orpeptide, the protein or peptide that protects a plant from a pathogen,the protein or peptide that enhances stress resistance in a plant, orthe plant binding protein or peptide.

Plant Growth Stimulating Proteins and Peptides

As noted above, the fusion proteins can comprise a targeting sequence,exosporium protein, or exosporium protein fragment and at least oneplant growth stimulating protein or peptide. For example, the plantgrowth stimulating protein or peptide can comprise a peptide hormone, anon-hormone peptide, an enzyme involved in the production or activationof a plant growth stimulating compound, or an enzyme that degrades ormodifies a bacterial, fungal, or plant nutrient source.

For example, where the plant growth stimulating protein or peptidecomprises a peptide hormone, the peptide hormone can comprise aphytosulfokine (e.g., phytosulfokine-α), clavata 3 (CLV3), systemin,Zm1GF, or a SCR/SP11.

Where the plant growth stimulating protein or peptide comprises anon-hormone peptide, the non-hormone peptide can comprise a RKN 16D10,Hg-Syv46, an eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS,or kunitz trypsin inhibitor (KTI).

The plant growth stimulating protein or peptide can comprise an enzymeinvolved in the production or activation of a plant growth stimulatingcompound. The enzyme involved in the production or activation of a plantgrowth stimulating compound can be any enzyme that catalyzes any step ina biological synthesis pathway for a compound that stimulates plantgrowth or alters plant structure, or any enzyme that catalyzes theconversion of an inactive or less active derivative of a compound thatstimulates plant growth or alters plant structure into an active or moreactive form of the compound.

The plant growth stimulating compound can comprise a compound producedby bacteria or fungi in the rhizosphere, e.g., 2,3-butanediol.

Alternatively, the plant growth stimulating compound can comprise aplant growth hormone, e.g., a cytokinin or a cytokinin derivative,ethylene, an auxin or an auxin derivative, a gibberellic acid or agibberellic acid derivative, abscisic acid or an abscisic acidderivative, or a jasmonic acid or a jasmonic acid derivative.

Where the plant growth stimulating compound comprises a cytokinin or acytokinin derivative, the cytokinin or the cytokinin derivative cancomprise kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine,dihydroxyzeatin, N6-(D2-isopentenyl) adenine, ribosylzeatin,N6-(D2-isopentenyl) adenosine, 2-methylthio-cis-ribosylzeatin,cis-ribosylzeatin, trans-ribosylzeatin,2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monosphosphate,N6-methylaminopurine, N6-dimethylaminopurine, 2′-deoxyzeatin riboside,4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin,meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopolin, ora combination thereof.

Where the plant growth stimulating compound comprises an auxin or anauxin derivative, the auxin or the auxin derivative can comprise anactive auxin, an inactive auxin, a conjugated auxin, a naturallyoccurring auxin, or a synthetic auxin, or a combination thereof. Forexample, the auxin or auxin derivative can comprise indole-3-aceticacid, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide,indole-3-acetonitrile, indole-3-ethanol, indole-3-pyruvate,indole-3-acetaldoxime, indole-3-butyric acid, a phenylacetic acid,4-chloroindole-3-acetic acid, a glucose-conjugated auxin, or acombination thereof.

The enzyme involved in the production or activation of a plant growthstimulating compound can comprise an acetoin reductase, anindole-3-acetamide hydrolase, a tryptophan monooxygenase, anacetolactate synthetase, an α-acetolactate decarboxylase, a pyruvatedecarboxylase, a diacetyl reductase, a butanediol dehydrogenase, anaminotransferase (e.g., tryptophan aminotransferase), a tryptophandecarboxylase, an amine oxidase, an indole-3-pyruvate decarboxylase, anindole-3-acetaldehyde dehydrogenase, a tryptophan side chain oxidase, anitrile hydrolase, a nitrilase, a peptidase, a protease, an adenosinephosphate isopentenyltransferase, a phosphatase, an adenosine kinase, anadenine phosphoribosyltransferase, CYP735A, a 5′ribonucleotidephosphohydrolase, an adenosine nucleosidase, a zeatin cis-transisomerase, a zeatin O-glucosyltransferase, a β-glucosidase, acis-hydroxylase, a CK cis-hydroxylase, a CK N-glucosyltransferase, a2,5-ribonucleotide phosphohydrolase, an adenosine nucleosidase, a purinenucleoside phosphorylase, a zeatin reductase, a hydroxylamine reductase,a 2-oxoglutarate dioxygenase, a gibberellic 2B/3B hydrolase, agibberellin 3-oxidase, a gibberellin 20-oxidase, a chitosinase, achitinase, a β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, anaminocyclopropane-1-carboxylic acid deaminase, or an enzyme involved inproducing a nod factor (e.g., nodA, nodB, or nodI).

Where the enzyme comprises a protease or peptidase, the protease orpeptidase can be a protease or peptidase that cleaves proteins,peptides, proproteins, or preproproteins to create a bioactive peptide.The bioactive peptide can be any peptide that exerts a biologicalactivity.

Examples of bioactive peptides include RKN 16D10 and RHPP.

The protease or peptidase that cleaves proteins, peptides, proproteins,or preproproteins to create a bioactive peptide can comprise subtilisin,an acid protease, an alkaline protease, a proteinase, an endopeptidase,an exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, acarboxylase, a serine protease, a glutamic protease, an aspartateprotease, a cysteine protease, a threonine protease, or ametalloprotease.

The protease or peptidase can cleave proteins in a protein-rich meal(e.g., soybean meal or yeast extract).

The plant growth stimulating protein can also comprise an enzyme thatdegrades or modifies a bacterial, fungal, or plant nutrient source. Suchenzymes include cellulases, lipases, lignin oxidases, proteases,glycoside hydrolases, phosphatases, nitrogenases, nucleases, amidases,nitrate reductases, nitrite reductases, amylases, ammonia oxidases,ligninases, glucosidases, phospholipases, phytases, pectinases,glucanases, sulfatases, ureases, xylanases, and siderophores. Whenintroduced into a plant growth medium or applied to a plant, seed, or anarea surrounding a plant or a plant seed, fusion proteins comprisingenzymes that degrade or modify a bacterial, fungal, or plant nutrientsource can aid in the processing of nutrients in the vicinity of theplant and result in enhanced uptake of nutrients by the plant or bybeneficial bacteria or fungi in the vicinity of the plant.

Suitable cellulases include endocellulases (e.g., an endogluconase suchas a Bacillus subtilis endoglucanase, a Bacillus thuringiensisendoglucanase, a Bacillus cereus endoglucanase, or a Bacillus clausiiendoglucanase), exocellulases (e.g., a Trichoderma reesei exocellulase),and β-glucosidases (e.g., a Bacillus subtilis β-glucosidase, a Bacillusthuringiensis β-glucosidase, a Bacillus cereus β-glucosidase, or aBacillus clausii β-glucosidase).

The lipase can comprise a Bacillus subtilis lipase, a Bacillusthuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausiilipase.

In one embodiment, the lipase comprises a Bacillus subtilis lipase. TheBacillus subtilis lipase can be PCR amplified using the followingprimers: ggatccatggctgaacacaatcc (forward, SEQ ID NO: 37) andggatccttaattcgtattctggcc (reverse, SEQ ID NO: 38).

In another embodiment, the cellulase is a Bacillus subtilisendoglucanase. The Bacillus subtilis endoglucanase can be PCR amplifiedusing the following primers: ggatccatgaaacggtcaatc (forward, SEQ ID NO:39) and ggatccttactaatttggttctgt (reverse, SEQ ID NO: 40).

In yet another embodiment, the fusion protein comprises an E. coliprotease PtrB. The E. coli protease PtrB can be PCR amplified using thefollowing primers: ggatccatgctaccaaaagcc (forward, SEQ ID NO: 41) andggatccttagtccgcaggcgtagc (reverse, SEQ ID NO: 42).

In certain embodiments, the fusion protein contains an endoglucanasewhich derives from the nucleotide sequence in SEQ ID NO: 104.

The amino acid sequence for an exemplary endoglucanase that may be fusedto the targeting sequence, an exosporium protein, or an exosporiumprotein fragment and, optionally, a linker sequence, such as a poly-Alinker, is the fusion protein provided as SEQ ID NO: 107.

In other embodiments, the fusion protein contains a phospholipase thatderives from the nucleotide sequence set forth in SEQ ID NO: 105.

The amino acid sequence for an exemplary phospholipase that may be fusedto the targeting sequence, an exosporium protein, or an exosporiumprotein fragment and, optionally, a linker sequence, such as a poly-Alinker, is the fusion protein provided as SEQ ID NO: 108.

In still other embodiments, the fusion protein contains a chitosanasethat derives from the nucleotide sequence set forth in SEQ ID NO: 106.The amino acid sequence for an exemplary chitosanase that may be fusedto the targeting sequence, an exosporium protein, or an exosporiumprotein fragment and, optionally, a linker sequence, such as a poly-Alinker, in the fusion protein is provided as SEQ ID NO: 109.

To create fusion constructs, genes may be fused to the native bc1Apromoter of Bacillus thuringiensis DNA encoding the first 35 amino acidsof Bc1A (amino acids 1-35 of SEQ ID NO: 1) using the splicing byoverlapping extension (SOE) technique. Correct amplicons are cloned intothe E. coli/Bacillus shuttle vector pIIP13, and correct clones screenedby DNA sequencing. Correct clones are electroporated into Bacillusthuringiensis (Cry-, plasmid-) and screened for chloramphenicolresistance. Correct transformants are grown in brain heart infusionbroth overnight at 30° C., plated onto nutrient agar plates, andincubated at 30° C. for 3 days. Spores expressing the fusion construct(BEMD spores) may be collected off of the plates by washing in phosphatebuffered saline (PBS) and purified by centrifugation and additionalwashes in PBS.

In such fusion proteins, the endoglucanase, phospholipase or chitosinasecan comprise a nucleotide sequence encoding an amino acid sequencehaving at least 85% identity with SEQ ID NO: 107, 108 or 109,respectively.

In such fusion proteins, the endoglucanase, phospholipase or chitosinasecan comprise an amino acid sequence having at least 90% identity withSEQ ID NO: 107, 108 or 109, respectively.

In such fusion proteins, the endoglucanase, phospholipase or chitosinasecan comprise an amino acid sequence having at least 95% identity withSEQ ID NO: 107, 108 or 109, respectively.

In such fusion proteins, the endoglucanase, phospholipase or chitosinasecan comprise an amino acid sequence having at least 98% identity withSEQ ID NO: 107, 108 or 109, respectively.

In such fusion proteins, the endoglucanase, phospholipase or chitosinasecan comprise an amino acid sequence having at least 99% identity withSEQ ID NO: 107, 108 or 109, respectively.

Suitable lignin oxidases comprise lignin peroxidases, laccases, glyoxaloxidases, ligninases, and manganese peroxidases.

The protease can comprise a subtilisin, an acid protease, an alkalineprotease, a proteinase, a peptidase, an endopeptidase, an exopeptidase,a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase,a serine protease, a glutamic protease, an aspartate protease, acysteine protease, a threonine protease, or a metalloprotease.

The phosphatase can comprise a phosphoric monoester hydrolase, aphosphomonoesterase (e.g., PhoA4), a phosphoric diester hydrolase, aphosphodiesterase, a triphosphoric monoester hydrolase, a phosphorylanhydride hydrolase, a pyrophosphatase, a phytase (e.g., Bacillussubtilis EE148 phytase or Bacillus thuringiensis BT013A phytase), atrimetaphosphatase, or a triphosphatase.

The nitrogenase can comprise a Nif family nitrogenase (e.g.,Paenibacillus massiliensis NifBDEHKNXV).

Proteins and Peptides that Protects Plants from Pathogens

The fusion proteins can comprise a targeting sequence, exosporiumprotein, or exosporium protein fragment, and at least one protein orpeptide that protects a plant from a pathogen.

The protein or peptide can comprise a protein or peptide that stimulatesa plant immune response. For example, the protein or peptide thatstimulates a plant immune response can comprise a plant immune systemenhancer protein or peptide. The plant immune system enhancer protein orpeptide can be any protein or peptide that has a beneficial effect onthe immune system of a plant. Suitable plant immune system enhancerproteins and peptides include harpins, α-elastins, β-elastins,systemins, phenylalanine ammonia-lyase, elicitins, defensins,cryptogeins, flagellin proteins, and flagellin peptides (e.g., flg22).

Alternatively, the protein or peptide that protects a plant from apathogen can be a protein or peptide that has antibacterial activity,antifungal activity, or both antibacterial and antifungal activity.Examples of such proteins and peptides include bacteriocins, lysozymes,lysozyme peptides (e.g., LysM), siderophores, non-ribosomal activepeptides, conalbumins, albumins, lactoferrins, lactoferrin peptides(e.g., LfcinB), streptavidin and TasA.

The protein or peptide that protects a plant from a pathogen can also bea protein or peptide that has insecticidal activity, helminthicidalactivity, suppresses insect or worm predation, or a combination thereof.For example, the protein or peptide that protects a plant from apathogen can comprise an insecticidal bacterial toxin (e.g., a VIPinsecticidal protein), an endotoxin, a Cry toxin (e.g., a Cry toxin fromBacillus thuringiensis), a protease inhibitor protein or peptide (e.g.,a trypsin inhibitor or an arrowhead protease inhibitor), a cysteineprotease, or a chitinase. Where the Cry toxin is a Cry toxin fromBacillus thuringiensis, the Cry toxin can be a Cry5B protein or a Cry21Aprotein. Cry5B and Cry21A have both insecticidal and nematocidalactivity.

The protein that protects a plant from a pathogen can comprise anenzyme. Suitable enzymes include proteases and lactonases. The proteasesand lactonases can be specific for a bacterial signaling molecule (e.g.,a bacterial lactone homoserine signaling molecule).

Where the enzyme is a lactonase, the lactonase can comprise1,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipateenol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase,gluconolactonase L-rhamnono-1,4-lactonase, limonin-D-ring-lactonase,steroid-lactonase, triacetate-lactonase, or xylono-1,4-lactonase.

The enzyme can also be an enzyme that is specific for a cellularcomponent of a bacterium or fungus. For example, the enzyme can comprisea β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, a chitosinase,a chitinase, a chitosinase-like enzyme, a lyticase, a peptidase, aproteinase, a protease (e.g., an alkaline protease, an acid protease, ora neutral protease), a mutanolysin, a stapholysin, or a lysozyme.

Proteins and Peptides that Enhance Stress Resistance in Plants

The fusion proteins can comprise a targeting sequence, exosporiumprotein, or exosporium protein fragment and at least one protein orpeptide that enhances stress resistance in a plant.

For example, the protein or peptide that enhances stress resistance in aplant comprises an enzyme that degrades a stress-related compound.Stress-related compounds include, but are not limited to,aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species,nitric oxide, oxylipins, and phenolics. Specific reactive oxygen speciesinclude hydroxyl, hydrogen peroxide, oxygen, and superoxide. The enzymethat degrades a stress-related compound can comprise a superoxidedismutase, an oxidase, a catalase, an aminocyclopropane-1-carboxylicacid deaminase, a peroxidase, an antioxidant enzyme, or an antioxidantpeptide.

The protein or peptide that enhances stress resistance in a plant canalso comprise a protein or peptide that protects a plant from anenvironmental stress. The environmental stress can comprise, forexample, drought, flood, heat, freezing, salt, heavy metals, low pH,high pH, or a combination thereof. For instance, the protein or peptidethat protects a plant from an environmental stress can comprises an icenucleation protein, a prolinase, a phenylalanine ammonia lyase, anisochorismate synthase, an isochorismate pyruvate lyase, or a cholinedehydrogenase.

Plant Binding Proteins and Peptides

The fusion proteins can comprise a targeting sequence, exosporiumprotein, or exosporium protein fragment and at least plant bindingprotein or peptide. The plant binding protein or peptide can be anyprotein or peptide that is capable of specifically or non-specificallybinding to any part of a plant (e.g., a plant root or an aerial portionof a plant such as a leaf, stem, flower, or fruit) or to plant matter.Thus, for example, the plant binding protein or peptide can be a rootbinding protein or peptide, or a leaf binding protein or peptide.

Suitable plant binding proteins and peptides include adhesins (e.g.,rhicadhesin), flagellins, omptins, lectins, expansins, biofilmstructural proteins (e.g., TasA or YuaB) pilus proteins, curlusproteins, intimins, invasins, agglutinins, and afimbrial proteins.

Recombinant Bacillus that Express the Fusion Proteins

The fusion proteins described herein can be expressed by recombinantexosporium-producing Bacillus cells. The fusion protein can be any ofthe fusion proteins discussed above.

The recombinant exosporium-producing Bacillus cells can coexpress two ormore of any of the fusion proteins discussed above. For example, therecombinant exosporium-producing Bacillus cells can coexpress at leastone fusion protein that comprises a plant binding protein or peptide,together with at least one fusion protein comprising a plant growthstimulating protein or peptide, at least one fusion protein comprising aprotein or peptide that protects a plant from a pathogen, or at leastone protein or peptide that enhances stress resistance in a plant.

The recombinant exosporium-producing Bacillus cells can compriseBacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillusmycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillusgaemokensis, Bacillus weihenstephensis, Bacillus toyoiensis or acombination thereof. For example, the recombinant exosporium-producingBacillus cells can comprise Bacillus cereus, Bacillus thuringiensis,Bacillus pseudomycoides, or Bacillus mycoides. In particular, therecombinant exosporium-producing Bacillus cells can comprise Bacillusthuringiensis or Bacillus mycoides.

To generate a recombinant exosporium-producing Bacillus cells expressinga fusion protein, any Bacillus cereus family member can be conjugated,transduced, or transformed with a vector encoding the fusion proteinusing standard methods known in the art (e.g., by electroporation). Thebacteria can then be screened to identify transformants by any methodknown in the art. For example, where the vector includes an antibioticresistance gene, the bacteria can be screened for antibiotic resistance.Alternatively, DNA encoding the fusion protein can be integrated intothe chromosomal DNA of a B. cereus family member host. The recombinantexosporium-producing Bacillus cells can then exposed to conditions whichwill induce sporulation. Suitable conditions for inducing sporulationare known in the art. For example, the recombinant exosporium-producingBacillus cells can be plated onto agar plates, and incubated at atemperature of about 30° C. for several days (e.g., 3 days).

Inactivated strains, non-toxic strains, or genetically manipulatedstrains of any of the above species can also suitably be used. Forexample, a Bacillus thuringiensis that lacks the Cry toxin can be used.Alternatively or in addition, once the recombinant B. cereus familyspores expressing the fusion protein have been generated, they can beinactivated to prevent further germination once in use. Any method forinactivating bacterial spores that is known in the art can be used.Suitable methods include, without limitation, heat treatment, gammairradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation,chemical treatment (e.g., treatment with gluteraldehyde, formaldehyde,hydrogen peroxide, acetic acid, bleach, or any combination thereof), ora combination thereof. Alternatively, spores derived from nontoxigenicstrains, or genetically or physically inactivated strains, can be used.

Recombinant Exosporium-Producing Bacillus Cells Having Plant-GrowthPromoting Effects and/or Other Beneficial Attributes

Many Bacillus cereus family member strains have inherent beneficialattributes. For example, some strains have plant-growth promotingeffects. Any of the fusion proteins described herein can be expressed insuch strains.

For example, the recombinant exosporium-producing Bacillus cells cancomprise a plant-growth promoting strain of bacteria.

The plant-growth promoting strain of bacteria can comprise a strain ofbacteria that produces an insecticidal toxin (e.g., a Cry toxin),produces a fungicidal compound (e.g., a β-1,3-glucanase, a chitosinase,a lyticase, or a combination thereof), produces a nematocidal compound(e.g., a Cry toxin), produces a bacteriocidal compound, is resistant toone or more antibiotics, comprises one or more freely replicatingplasmids, binds to plant roots, colonizes plant roots, forms biofilms,solubilizes nutrients, secretes organic acids, or any combinationthereof.

For example, where the recombinant exosporium-producing Bacillus cellscomprises a plant-growth promoting strain of bacteria, the plantgrowth-promoting strain of bacteria can comprise Bacillus mycoides BT155(NRRL No. B-50921), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillusmycoides EE141 (NRRL No. B-50916), Bacillus mycoides BT46-3 (NRRL No.B-50922), Bacillus cereus family member EE128 (NRRL No. B-50917),Bacillus thuringiensis BT013A (NRRL No. B-50924), or Bacillus cereusfamily member EE349 (NRRL No. B-50928). Bacillus thuringiensis BT013A isalso known as Bacillus thuringiensis 4Q7. Each of these strains wasdeposited with the United States Department of Agriculture (USDA)Agricultural Research Service (ARS), having the address 1815 NorthUniversity Street, Peoria, Ill. 61604, U.S.A., on Mar. 10, 2014, and isidentified by the NRRL deposit number provided in parentheses.

These plant-growth promoting strains were isolated from the rhizospheresof various vigorous plants and were identified by their 16S rRNAsequences, and through biochemical assays. The strains were identifiedat least to their genus designation by means of conventionalbiochemistry and morphological indicators. Biochemical assays forconfirmed Gram-positive strains such as Bacillus included growth on PEAmedium and nutrient agar, microscopic examination, growth on 5% and 7.5%NaCl medium, growth at pH 5 and pH 9, growth at 42° C. and 50° C., theability to produce acid upon fermentation with cellobiose, lactose,glycerol, glucose, sucrose, d-mannitol, and starch; fluorescent pigmentproduction; gelatin hydrolysis; nitrate reduction; catalase production,starch hydrolysis; oxidase reaction, urease production and motility.

For example, the recombinant exosporium-producing Bacillus cellscomprising a plant-growth promoting strain of bacteria can compriseBacillus mycoides BT155, Bacillus mycoides EE141, or Bacillusthuringiensis BT013A. The recombinant exosporium-producing Bacilluscells can express any of the fusion proteins described herein, e.g., afusion protein comprising the targeting sequence of SEQ ID NO: 60 and anon-hormone peptide (e.g., kunitz trypsin inhibitor (KTI)), an enzymeinvolved in the production or activation of a plant growth stimulatingcompound (e.g., a chitosinase), a plant binding protein or peptide(e.g., TasA); a protein or peptide that protects a plant from a pathogen(e.g., TasA), or an enzyme that degrades or modifies a bacterial,fungal, or plant nutrient source (e.g., a phosphatase such as PhoA orphytase, or an endoglucanase).

Promoters

In any of the recombinant exosporium-producing Bacillus cells describedherein, the fusion protein can be expressed under the control of apromoter that is native to the targeting sequence, the exosporiumprotein, or the exosporium protein fragment of the fusion protein. Forexample, where the fusion protein comprises a targeting sequence derivedfrom B. anthracis Sterne Bc1A (e.g., amino acids 20-35 of SEQ ID NO: 1,amino acids 1-35 of SEQ ID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 60) orwhere the fusion protein comprises full length Bc1A (SEQ ID NO: 2) or afragment of full length Bc1A (e.g., SEQ ID NO: 59), the fusion proteincan be expressed under the control of a promoter that is normallyassociated with the Bc1A gene in the genome of B. anthracis Sterne(e.g., the promoter of SEQ ID NO: 85).

Alternatively, the fusion protein can be expressed under the control ofa high-expression sporulation promoter. In some cases, the promoter thatis native to the targeting sequence, exosporium protein, or exosporiumprotein fragment will be a high-expression sporulation promoter. Inother cases, the promoter that is native to the targeting sequence,exosporium protein, or exosporium protein fragment will not be ahigh-expression sporulation promoter. In the latter cases, it may beadvantageous to replace the native promoter with a high-expressionsporulation promoter. Expression of the fusion protein under the controlof a high-expression sporulation promoter provides for increasedexpression of the fusion protein on the exosporium of the Bacilluscereus family member.

The high-expression sporulation promoter can comprise one or moresigma-K sporulation-specific polymerase promoter sequences.

Suitable high-expression sporulation promoters for use in expressing thefusion proteins in a Bacillus cereus family member include those listedin Table 2 below:

TABLE 2 Promoter Sequences Promoter (SEQ ID NO.) Sequence BclA promoterTAATCACCCTCTTCCAAATCAAT CATATGT (B. anthracis TA TA CATATACTAAACTTTCCATTTTTTTA Sterne) AATTGTTCAAGTAGTTTAAGATTTCTTTTC (SEQ ID NO: 85)AATAATTCAAATGTCCGTGTCATTTTCTTT CGGTTTTG CATCTACTA TATAATGAACGCTTTATGGAGGTGAATTTATG BetA promoter ATTTATTTCATTCAATTTTTCCTATTTAGT(B. anthracis ACCTACCGCACTCACAAAAAGCACCTCTCA Sterne)TTAATTTATATTATAGTCATTGAAATCTAA (SEQ ID NO: 86) TTTAATGAAATCAT CATACTATATGTTTTA TAAGAAGTAAAGGTAC CATACTTAA TTAAT ACATATCTATACACTTCAATATCACAGCATGCAGTTGAATTATATCCAACTTTCATTTCA AATTAAATAAGTGCCTCCGCTATTGTGAATGTCATTTACTCTCCCTACTA CATTTAATA A TTATGACAAGCAATCATAGGAGGTTACTAC ATGBAS1882 AATTACATAACAAGAACTACATTAGGGAGC promoterAAGCAGTCTAGCGAAAGCTAACTGCTTTTT (B. anthracisTATTAAATAACTATTTTATTAAATTTCATA Sterne) TATACAATCGCTTGTCCATTTCATTTGGCT(SEQ ID NO: 87) CTACCCACG CATTTACTA TTAGTAATATGA ATTTTTCAGAGGTGGATTTTATTGene 3572 CTATGATTTAAGATACACAATAGCAAAAGA promoter GAAA CATATTATATAACGATAAATGAAACT (B. TATGTATATGTATGGTAACTGTATATATTA weihenstephensisCTACAATACAGTATACTCATAGGAGGTAGG KBAB 4) TATG (SEQ ID NO: 88) YVTN β-GGTAGGTAGATTTGAAATATGATGAAGAAA propeller proteinAGGAATAACTAAAAGGAGTCGATATCCGAC promoter TCCTTTTAGTTATAAATAATGTGGAATTAG(B. AGTATAATTTTATATAGGTATATTGTATTA weihenstephensisGATGAACGCTTTATCCTTTAATTGTGATTA KBAB 4) ATGATGGATTGTAAGAGAAGGGGCTTACAG(SEQ ID NO: 89) TCCTTTTTTTATGGTGTTCTATAAGCCTTTTTAAAAGGGGTACCACCCCACACCCAAAAA CAGGGGGGGTTATAACTACATATTGGATGTTTTGTAACGTACAAGAATCGGTATTAATTA CCCTGTAAATAAGTTATGTGTATATAAGGT AACTTTATATATTC TCCTACAATAAAATAA AGGAGGTAATAAAGTG Cry1A promoterAACCCTTAATGCATTGGTTAAACATTGTAA (B. thuringiensisAGTCTAAAGCATGGATAATGGGCGAGAAGT HD-73) AAGTAGATTGTTAACACCCTGGGTCAAAAA(SEQ ID NO: 90) TTGATATTTAGTAAAATTAGTTGCACTTTG TGCATTTTTT CATAAGATG AGTCATATGTT T TAAATTGTAGTAATGAAAAACAGTATTAT ATCATAATGA ATTGGTATCTTAATAAAAGA GATGGAGGTAACTTA ExsY promoterTAATTCCACCTTCCCTTATCCTCTTTCGCC (B. thuringiensisTATTTAAAAAAAGGTCTTGAGATTGTGACC serovar AAATCTCCTCAACTCC AATATCTTA TTAATkonkukian str. GTAAATACAAACAAGAAGATAAGGAGTGAC 97-27) ATTAA(SEQ ID NO: 91) CotY promoter AGGATGTCTTTTTTTATATTGTATTATGTA(B. thuringiensis CATCCCTACTATATAAATTCCCTGCTTTTA Al Hakam)TCGTAAGAATTAACGTAATATCAACCATAT (SEQ ID NO: 92) CCCGTT CATATTGTAGTAGTGTATGTCAGA ACTCACGAGAAGGAGTGAACATAA YjcA promoterTTAATGTCACTCCTTATCTTCTTGTTTGTA (B. thuringiensis TTTACATT AATAAGATATTGGAGTTGAGGA serovar kurstaki GATTTGGTCACAATCTCAAGACCTTTTTTT str. HD73)TAAATAGGCGAAAGAGGATAAGGGAAGGTG (SEQ ID NO: 93) GAATTA YjcB promoterATATATTTTCATAATACGAGAAAAAGCGGA (B. thuringiensisGTTTAAAAGAATGAGGGAACGGAAATAAAG serovar kurstaki AGTTGTT CATATAGTAAATAGACAGAATTG str. HD73) ACAGTAGAGGAGA (SEQ ID NO: 94) BxpB promoterAAACTAAATAATGAGCTAAGCATGGATTGG (B. thuringiensisGTGGCAGAATTATCTGCCACCCAATC CATG Al Hakam) CTTAACGAGTATTATTATGTAAATTTCTTA (SEQ ID NO: 95) AAATTGGGAACTTGTCTAGAACATAGAACCTGTCCTTTT CATTAACTG AAAGTAGAAACA GATAAAGGAGTGAAAAACA RhamnoseATTCACTACAACGGGGATGAGTTTGATGCG promoter (B. GATA CATATGAGAAGTACCGGAAAGTGTTT thuringiensis GTAGAA CATTACAA AGATATATTATCTCCAAl Hakam) TCATAAAGGAGAGATGCAAAG (SEQ ID NO: 96) CotY/CotZCGCGCACCACTTCGTCGTACAACAACGCAA promoter (B.GAAGAAGTTGGGGATACAGCAGTATTCTTA anthracis TTCAGTGATTTAGCACGCGGCGTAACAGGASterne) GAAAACATTCACGTTGATTCAGGGTAT CAT (SEQ ID NO: 97) ATCTTAGGATAAATATAATATTAATTTTAA AGGACAATCTCTACATGTTGAGATTGTCCTTTTTATTTGTTCTTAGAAAGAACGATTTTT AACGAAAGTTCTTACCACGTTATGAATATAAGTATAATAGTACACGATTTATTCAGCTAC GTA BclC promoterTGAAGTATCTAGAGCTAATTTACGCAAAGG (B. anthracisAATCTCAGGACAACACTTTCGCAACACCTA Sterne) TATTTTAAATTTAATAAAAAAAGAGACTCC(SEQ ID NO: 98) GGAGTCAGAAATTATAAAGCTAGCTGGGTTCAAATCAAAAATTTCACTAAAACGATATTA TCAATACGCAGAAAATGGAAAAAACGCCTTATCATAAGGCGTTTTTTCCATTTTTTCTTC AAACAAACGATTTTACTATGACCATTTAAC TAATTTTTGCATCTACTA TGATGAGTTTCA TTCACATTCTCATTAGAAAGGAGAGATTTA ATG Sigma KTATATCATATGTAAAATTAGTTCTTATTCC promoter CA CATATCATA TAGAATCGC CATATTATAC (B. anthracis ATGCAGAAAACTAAGTATGGTATTATTCTT Sterne)AAATTGTTTAGCACCTTCTAATATTACAGA (SEQ ID NO: 99)TAGAATCCGTCATTTTCAACAGTGAACATG GATTTCTTCTGAACACAACTCTTTTTCTTTCCTTATTTCCAAAAAGAAAAGCAGCCCATT TTAAAATACGGCTGCTTGTAATGTACATTAInhA promoter TATCACATAACTCTTTATTTTTAATATTTC (B. thuringiensis GACATAAAGTG AAACTTTAATCAGTGGGGG Al Hakam) CTTTGTTCATCCCCCCACTGATTATTAATT(SEQ ID NO: 100) GAACCAAGGGATAAAAAGATAGAGGGTCTGACCAGAAAACTGGAGGGCATGATTCTATAA CAAAAAGCTTAATGTTTATAGAATTATGTCTTTTTATATAGGGAGGGTAGTAAACAGAGA TTTGGACAAAAATGCACCGATTTATCTGAATTTTAAGTTTTATAAAGGGGAGAAATG BclA cluster ATTTTTTACTTAGCAGTAAAACTGATATCAglycosyl GTTTTACTGCTTTTTCATTTTTAAATTCAA transferaseTCATTAAATCTTCCTTTTCTACATAGT CAT operon 1 AATGTT GTATGACATTCCGTAGGAGGCACT(B. thuringiensis TATA serovar konkukian str. 97-27) (SEQ ID NO: 101)BclA cluster ACATAAATTCACCTCCATAAAGCGTTCATT glycosylATATAGTAGATGCAAAACCGAAAGAAAATG transferaseACACGGACATTTGAATTATTGAAAAGAAAT operon 2 CTTAAACTACTTGAACAATTTAAAAAAATG(B. thuringiensis GAAAGTTTAGTATATGTATAA CATATGATT serovar kurstakiGATTTGGAAGAGGGTGATTA str. HD73) (SEQ ID NO: 102) Glycosyl TTCTATTTTCCAACATAACATG CTACGATT transferase AAATGGTTTTTTGCAAATGCCTTCTTGGGA promoterAGAAGGATTAGAGCGTTTTTTTATAGAAAC (B. thuringiensisCAAAAGTCATTAACAATTTTAAGTTAATGA Al Hakam) CTTTTTTGTTTGCCTTTAAGAGGTTTTATG(SEQ ID NO: 103) TTACTATAATTATAGTATCAGGTACTAATAACAAGTATAAGTATTTCTGGGAGGATATAT CA

In the promoter sequences listed in Table 2 above, the locations of thesigma-K sporulation-specific polymerase promoter sequences are indicatedby bold and underlined text. The Cry1A promoter (B. thuringiensis HD-73;SEQ ID NO: 90) has a total of four sigma-K sequences, two of whichoverlap with one another, as indicated by the double underlining inTable 2.

Preferred high-expression sporulation promoters for use in expressingthe fusion proteins in a Bacillus cereus family member include the BetApromoter (B. anthracis Sterne; SEQ ID NO: 86), the Bc1A promoter (B.anthracis Sterne; SEQ ID NO: 85), the Bc1A cluster glycosyl transferaseoperons 1 and 2 promoters (B. anthracis Sterne; SEQ ID NOS: 101 and102), and the YVTN β-propeller protein promoter (B. weihenstephensisKBAB 4; SEQ ID NO: 89).

In any of the recombinant exosporium-producing Bacillus cells describedherein, the fusion protein can be expressed under the control of asporulation promoter comprising a nucleic acid sequence having at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identity with a nucleic acid sequence of any one of SEQ ID NOS: 85-103.

When the sporulation promoter comprising a nucleic acid sequence havingat least 80%, at least 90%, at least 95%, at least 98%, or at least 99%identity with a nucleic acid sequence of any one of SEQ ID NOS: 85-103,the sigma-K sporulation-specific polymerase promoter sequence orsequences preferably have 100% identity with the correspondingnucleotides of SEQ ID NO: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, or 103. For example, as illustrated inTable 2 above, the Bc1A promoter of B. anthracis Sterne (SEQ ID NO: 85)has sigma-K sporulation-specific polymerase promoter sequences atnucleotides 24-32, 35-43, and 129-137. Thus, if the sporulation promotercomprises a sequence having at least 90% identity with the nucleic acidsequence of SEQ ID NO: 85, it is preferred that the nucleotides of thesporulation promoter corresponding to nucleotides 24-32, 35-43, and129-137 of SEQ ID NO: 85 have 100% identity with nucleotides 24-32,35-43, and 129-137 of SEQ ID NO: 85.

In any of the methods described herein for stimulating plant growth,plants grown in the plant growth medium comprising the recombinantexosporium-producing Bacillus cells and at least one further biologicalcontrol agent selected from the particular microorganisms disclosedherein exhibit increased growth as compared to the growth of plants inthe identical plant growth medium that does not contain the recombinantexosporium-producing Bacillus cells.

In any of the compositions and methods described herein for stimulatingplant growth, the recombinant exosporium-producing Bacillus cells cancomprise any of the recombinant plant-growth promoting strains ofbacteria described above.

In any of the compositions or methods for stimulating plant growthdisclosed herein, the fusion protein can be expressed under the controlof any of the promoters described above.

Synthetic Nodulation Factors and Plant Growth Stimulators

In some embodiments, the compositions comprising recombinantexosporium-producing Bacillus cells that express a fusion protein and atleast one of the biological control agents described herein furthercomprise a synthetic nodulation factor and/or plant growth stimulator. Anon-limiting example of such a synthetic compound is a compound ofgeneral formula (I)

wherein:

-   -   n is 2 or 3;    -   A is —C(O)—;    -   B is a phenylene;    -   C is —O—;    -   D is a linear hydrocarbon-based chain comprising 11 carbons,        which is saturated or unsaturated between carbons 4 and 5;    -   E and G are independently selected from the group consisting of        a substituent NHR20;    -   R1, R2, R3, R4, R5, R6, R7, and R9 are H;    -   R8 is selected from the group consisting of H, fucosyl,        methylfucosyl, SO₃H, SO₃Li, SO₃Na, SO₃K, and SO₃N(C₁₋₈-alkyl)₄;    -   R20 is C(O)C₁₋₆-alkyl; and    -   any agriculturally acceptable geometrical and/or optical isomer,        enantiomer and/or diastereoisomer, tautomer, salt, N-oxide,        sulfoxide, or sulfone thereof.

The salt may be selected from the group consisting of lithium, sodium,potassium, and tetraalkylammonium salts.

In certain embodiments, E and G are NHC(O)CH₃.

In other embodiments, R8 is selected from the group consisting of H,SO₃H, SO₃Li, SO₃Na, SO₃K, SO₃N(C₁₋₈alkyl)₄ and a substituent of formula:

wherein:

-   -   R26 is selected from the group consisting of H and CH₃; and    -   R27 and R28 are independently selected from the group consisting        of H, C(O)CH₃, SO₃H, SO₃Li, SO₃Na, SO₃K and SO₃N(C₁₋₈alkyl)₄.

In some aspects, R26, R27, and R28 are all hydrogen.

Additional non-limiting examples of a synthetic nodulation factor and/orplant growth stimulator that can be used in the present inventioninclude compounds of the structural formulae:

-   -   in which, when it is present, M is selected from the group        consisting of H⁺, Li⁺, Na⁺, K⁺ and (C₁₋₈alkyl)₄N⁺.

In certain aspects, the synthetic nodulation factor and/or plant growthstimulator is a compound selected from the group consisting of:

Additional Biological Control Agents

Biological control agents can include, in particular, bacteria, fungi oryeasts, protozoa, viruses, entomopathogenic nematodes, inoculants andbotanicals and/or mutants of them having all identifying characteristicsof the respective strain, and/or at least one metabolite produced by therespective strain that exhibits activity against insects, mites,nematodes and/or phytopathogens. The present invention relates to thecombinations of the above-described recombinant Bacillus cells with theparticular biological control agents described herein and/or to mutantsof specific strains of microorganisms described herein, where themutants have all the identifying characteristics of the respectivestrain, and/or at least one metabolite produced by the respective strainthat exhibits activity against insects, mites, nematodes and/orphytopathogens or promotes plant growth and/or enhances plant health.

The bacterial cells, spores and metabolites in culture broth resultingfrom fermentation (the “whole broth” or “fermentation broth”) of theparticular microorganisms described herein may be used directly orconcentrated by conventional industrial methods, such as centrifugation,filtration, and evaporation, or processed into dry powder and granulesby spray drying, drum drying and freeze drying, for example.

The terms “whole broth” and “fermentation broth,” as used herein, referto the culture broth resulting from fermentation before any downstreamtreatment. The whole broth encompasses the microorganism and itscomponent parts, unused raw substrates, and metabolites produced by themicroorganism during fermentation. The term “broth concentrate,” as usedherein, refers to whole broth (fermentation broth) that has beenconcentrated by conventional industrial methods, as described above, butremains in liquid form. The term “fermentation solid,” as used herein,refers to dried fermentation broth. The term “fermentation product,” asused herein, refers to whole broth, broth concentrate and/orfermentation solids. Compositions of the present invention includefermentation products. In some embodiments, the concentratedfermentation broth is washed, for example, via a diafiltration process,to remove residual fermentation broth and metabolites.

In another embodiment, the fermentation broth or broth concentrate canbe dried with or without the addition of carriers, inerts, or additivesusing conventional drying processes or methods such as spray drying,freeze drying, tray drying, fluidized-bed drying, drum drying, orevaporation.

According to the invention, biological control agents, which aresummarized under the term “bacteria”, include spore-forming,root-colonizing bacteria, or bacteria and their metabolites useful asbiological insecticides, -nematicides, miticides, or -fungicide or soilamendments improving plant health and growth. Bacteria to be used oremployed according to the invention follow.

B. cereus strains, including strain CNCM 1-1562 (cf. U.S. Pat. No.6,406,690), Bacillus firmus, Bacillus pumilus, in particular strain GB34(products known as YIELD SHIELD®), and strain QST2808 (products known asSONATA® QST2808) Bacillus subtilis, in particular strain GB03 (productsknown as KODIAK®), strain QST713 (products known as SERENADE® QST713),strain AQ30002 (aka QST30002; NRRL Accession No. B-50421, known from WO2012/087980, which is incorporated herein by reference), strain AQ30004(aka QST30004; NRRL Accession No. B-50455, known from WO 2012/087980,which is incorporated herein by reference), strain AQ743 (NRRL AccessionNo. B-21665), strain AQ153 (ATCC Accession No. 55614 as described in WO98/21964), (1.79) Streptomyces galbus strain AQ 6047 (Accession NumberNRRL 30232), (1.91) Rhodococcus globerulus AQ719 (NRRL Accession No.B-21663), (1.92) Bacillus sp. AQ175 (ATCC Accession No. 55608), (1.93)Bacillus sp. AQ 177 (ATCC Accession No. 55609), (1.94) Bacillus sp.AQ178 (ATCC Accession No. 53522), (1.95) Streptomyces sp. straindescribed in WO 02/26041 A2 (NRRL Accession No. B-30145), (1.96)Streptomyces microflavus strain NRRL B-50550, (1.97) Streptomycesmicroflavus strain M (Accession No. 091013-02), gougerotin-containingfermentation products of Streptomyces, as described in WO2014/059275,and Streptomyces galbus QST6047, described in U.S. Pat. No. 6,682,925.

In a particular embodiment the following bacteria are used incombination with the exosporium-producing recombinant Bacillus cellsdescribed above:

Bacillus firmus, in particular strain 1-1582 (products known as Bionem,Votivo, Flocter),

Bacillus pumilus, in particular strain GB34 (products known as YIELDSHIELD®), and strain QST2808 (products known as SONATA® QST2808),

Bacillus subtilis, in particular strain GB03 (products known asKODIAK®), strain QST713 (products known as SERENADE® QST713); NRRLAccession No. B-50455, known from WO 2012/087980, which is incorporatedherein by reference), or B. subtilis var. amyloliquefaciens strain FZB24(products known as TAEGRO®), strain AQ743 (NRRL Accession No. B-21665),strain AQ153 (ATCC Accession No. 55614 as described in WO 98/21964),

In one embodiment the composition of the present invention comprises acombination of at least one of the particular biological control agentsdescribed herein and at least one further biological control agentselected from the group consisting of Bacillus chitinosporus AQ746 (NRRLAccession No. B-21618), Bacillus mycoides AQ726 (NRRL Accession No.B-21664), Bacillus pumilus QST2808 (NRRL Accession No. B-30087),Bacillus pumilus AQ717 (NRRL Accession No. B-21662), Bacillus sp. AQ175(ATCC Accession No. 55608), Bacillus sp. AQ177 (ATCC Accession No.55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), Bacillus subtilisAQ743 (NRRL Accession No. B-21665), Bacillus subtilis AQ713 (NRRLAccession No. B-21661), Bacillus subtilis AQ153 (ATCC Accession No.55614), Muscodor albus 620 (NRRL Accession No. 30547), Muscodor roseusA3-5 (NRRL Accession No. 30548), Rhodococcus globerulus AQ719 (NRRLAccession No. B-21663), Streptomyces galbus (NRRL Accession No. 30232),Streptomyces sp. (NRRL Accession No. B-30145), Bacillus subtilis AQ30002(NRRL Accession No. B-50421), and Bacillus subtilis AQ30004 (NRRLAccession No. B-50455) and/or a mutant of these strains having all theidentifying characteristics of the respective strain, and/or at leastone metabolite produced by the respective strain that exhibits activityagainst insects, mites, nematodes and/or phytopathogens.

Said further biological control agents are known in the art as follows:

Bacillus chitinosporus AQ746 (NRRL Accession No. B-21618) is known fromWO 98/21966 A2. It is specifically active against nematodes and insectsand produces non-exotoxin, non-proteinaceous, active metabolites in itssupernatant. Those metabolites are active against nematodes andcockroaches, but inactive against flies, corn rootworm or beet armyworm.

Bacillus mycoides AQ726 (NRRL Accession No. B-21664) and itswater-soluble metabolites kill or stunt insects such as corn rootwormlarvae and aphids (WO 99/09820 A1).

As described in WO 00/58442 A1 Bacillus pumilus QST2808 (NRRL AccessionNo. B-30087) is able to inhibit a broad range of fungal plant diseasesin vivo. Commercially available formulations of this strain are soldunder the tradenames SONATA® and BALLAD® PLUS from Bayer CropScience LP(North Carolina, USA).

Bacillus pumilus AQ717 (NRRL Accession B-21662) is known from WO99/10477 A1. It produces a metabolite that exhibits pesticidal activityagainst corn rootworms, nematodes and beet armyworms.

The bacterial strains Bacillus sp. AQ175 (ATCC Accession No. 55608),Bacillus sp. AQ177 (ATCC Accession No. 55609) and Bacillus sp. AQ178(ATCC Accession No. 53522) described in WO 98/21967 A1 are effective intreating and protecting plants from aboveground fungal and bacterialinfections.

The metabolite-producing strain Bacillus subtilis AQ743 (NRRL AccessionNo. B-21665) kills or stunts corn rootworm larvae, beet armyworm larvae,fly adults and nematodes (cf. WO 99/09819).

Bacillus subtilis AQ713 (Accession No. B-21661), also named Bacillussubtilis QST713, exhibits broad fungicidal and bactericidal activity andalso exhibits corn rootworm activity (WO 98/50422 A1). Commerciallyavailable formulation of this strain are available under the tradenamesSERENADE® MAX, SERENADE SOIL®, SERENADE® ASO, SERENADE® CPB andRHAPSODY® from Bayer CropScience LP (North Carolina, USA). The SERENADE®product (U.S. EPA Registration No. 69592-12) contains a patented strainof Bacillus subtilis (strain QST713) and many different lipopeptidesthat work synergistically to destroy disease pathogens and providesuperior antimicrobial activity. The SERENADE® product is used toprotect plants such as vegetables, fruit, nut and vine crops againstdiseases such as Fire Blight, Botrytis, Sour Rot, Rust, Sclerotinia,Powdery Mildew, Bacterial Spot and White Mold. The SERENADE® productsare available as either liquid or dry formulations which can be appliedas a foliar and/or soil treatment. Copies of U.S. EPA Master Labels forthe SERENADE® products, including SERENADE® ASO, SERENADE® MAX, andSERENADE SOIL®, are publicly available through National PesticideInformation Retrieval System's (NPIRSv) USEPA/OPP Pesticide ProductLabel System (PPLS).

SERENADE® ASO (Aqueous Suspension-Organic) contains 1.34% of driedQST713 as an active ingredient and 98.66% of other ingredients.SERENADE® ASO is formulated to contain a minimum of 1×10⁹ cfu/g ofQST713 while the maximum amount of QST713 has been determined to be3.3×10¹⁰ cfu/g. For further information, see the U.S. EPA Master Labelsfor SERENADE® ASO dated Jan. 4, 2010, which is incorporated by referenceherein in its entirety.

SERENADE® MAX contains 14.6% of dried QST713 as an active ingredient and85.4% of other ingredients. SERENADE® MAX is formulated to contain aminimum of 7.3×10⁹ cfu/g of QST713 while the maximum amount of QST713has been determined to be 7.9×10¹⁰ cfu/g. For further information, seethe U.S. EPA Master Label for SERENADE® MAX, which is incorporated byreference herein in its entirety.

Bacillus subtilis AQ153 (ATCC Accession No. 55614) as described in WO98/21964 A1 is effective in inhibiting growth of plant pathogenicbacteria and fungi.

WO 02/02082898 A1 describes endophytic fungi including Muscodor albus620, also known as Muscodor albus QST20799 (NRRL Accession No. 30547)and Muscodor roseus A3-5 (NRRL Accession No. 30548) that produce amixture of volatile antibiotics with activity against fungi, bacteria,insects and nematodes.

Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663) producesmetabolites that exhibits pesticidal activity against corn rootworms(U.S. Pat. No. 6,027,723 A).

WO 01/79480 A2 describes a strain of Streptomyces galbus (NRRL AccessionNo. 30232) which shows insecticidal activity against Lepidoptera.

The Streptomyces sp. strain described in WO 02/26041 A2 (NRRL AccessionNo. B-30145) exhibits antifungal activity on specific plant pathogenssuch as Alternaria, Phytophthora, Botrytis, Rhizoctonia and Sclerotinia.

The strains Bacillus subtilis AQ30002 (also known as QST30002) (NRRLAccession No. B-50421, deposited on Oct. 5, 2010) and Bacillus subtilisAQ30004 (also known as QST30004) (NRRL Accession No. B-50455, depositedon October 5, 2010) are known from WO 2012/087980 A1, which isincorporated herein by reference. As described therein, these BCAsexhibit a broad fungicidal and bactericidal activity. B19 and B20 have amutation in the swrA gene that results in impaired swarming ability andenhanced plant health promotion compared to a strain containing awildtype swrA gene. The mutation causes these BCAs to form a more robustbiofilm than the wildtype strain, thereby enhancing its fungicidal andbactericidal activity.

In some embodiments, the biological control agent is a Bacillus subtilisstrain, such as Bacillus subtilis QST713, which produces a fengycin-typecompound, an iturin-type compound, and/or a surfactin-type compound. Insome aspects, the lipopeptide is a fengycin-type compound such asplipastatin A1, plipastatin B1, plipastatin B2, fengycin A, fengycin B,agrastatin 1, or agrastatin 2. In other aspects, the lipopeptide is aniturin-type compound such as iturin A, mycosubtilin, or bacillomycin.

Other lipopeptide-producing strains that are suitable for use in thecompositions and methods of the present invention include Bacillusamyloliquefaciens strain D747 (available as BACSTAR® from Etec CropSolutions, NZ and also available as DOUBLE NICKEL™ from Certis, US);Bacillus subtilis MBI600 (available as SUBTILEX® from Becker Underwood,U.S. EPA Reg. No. 71840-8); Bacillus subtilis Y1336 (available asBIOBAC® WP from Bion-Tech, Taiwan, registered as a biological fungicidein Taiwan under Registration Nos. 4764, 5454, 5096 and 5277); Bacillusamyloliquefaciens, in particular strain FZB42 (available as RHIZOVITAL®from ABiTEP, DE); and Bacillus subtilis var. amyloliquefaciens FZB24 isavailable from Novozymes Biologicals Inc. (Salem, Va.) or Syngenta CropProtection, LLC (Greensboro, N.C.) as the fungicide TAEGRO® or TAEGRO®ECO (EPA Registration No. 70127-5).

In some embodiments, the biological control agent in the synergisticcombinations of the present invention is:

Bacillus firmus, including strain 1-1582 (products known as Bionem,Votivo, Flocter), disclosed in U.S. Pat. No. 6,406,690 (which is hereinincorporated by reference) and deposited with the CNCM on May 29, 1995,with Accession No. CNCM I-1582,

Bacillus pumilus, including strain GB34 (products known as YIELDSHIELD®) and strain QST2808 (products known as SONATA® QST2808),

Bacillus subtilis and Bacillus amyloliquefaciens, including those thatproduce lipopeptides and, in particular, a combination of plipastatinsor fengycins, surfactins and/or iturins. Also, as to Bacillus subtilis,in particular strain GB03 (products known as KODIAK®, c.f. U.S. EPA,Pesticide Fact Sheet—Bacillus subtilis GB03 1992), strain QST713(products known as SERENADE® QST713), strain AQ30002 (aka QST30002; NRRLAccession No. B-50421, known from WO 2012/087980, which is incorporatedherein by reference), and strain AQ30004 (aka QST30004; NRRL AccessionNo. B-50455, known from WO 2012/087980, which is incorporated herein byreference).

According to the invention biological control agents which may becomprised in the composition of the invention and that are summarizedunder the term “fungi” or “yeasts” are the following organisms andand/or mutants of them having all identifying characteristics of therespective strain, and/or metabolites produced by the respective strainthat exhibit activity against insects, mites, nematodes and/orphytopathogens (the numbering is used in the complete description):

Muscodor albus, in particular strain QST20799 (products known asARABESQUE™ or ANDANTE™), Coniothyrium minitans, in particular strainCON/M/91-8 (products known as CONTANS®), Lagenidium giganteum (productsknown as LAGINEX® by AgraQuest, Inc.), Paecilomyces lilacinus, inparticular spores of P. lilacinus strain 251 (products known as BIOACT®,cf. Crop Protection 2008, 27, 352-361).

According to one embodiment of the present invention the biologicalcontrol agent comprises not only the pure cultures of the respectivemicroorganisms, but also their suspensions in a whole broth culture or ametabolite-containing supernatant or a purified metabolite obtained fromwhole broth culture of the strain. “Whole broth culture” refers to aliquid culture containing both cells and media. “Supernatant” refers tothe liquid broth remaining when cells grown in broth are removed bycentrifugation, filtration, sedimentation, or other means well known inthe art.

The above-mentioned metabolites produced by the nonpathogenicmicroorganisms include antibiotics, enzymes, siderophores and growthpromoting agents, for example zwittermicin-A, kanosamine, polyoxine,enzymes such as α-amylase, chitinases, and pektinases, phytohormones andprecursors thereof, such as auxines, gibberlin-like substances,cytokinin-like compounds, lipopeptides such as iturins, plipastatins orsurfactins, e.g., agrastatin A, bacillomycin D, bacilysin, difficidin,macrolactin, fengycin, bacilysin and bacillaene.

According to the invention, the biological control agents describedherein may be employed or used in any physiologic state such as activeor dormant.

Compositions According to the Present Invention

According to the present invention the composition comprises a) arecombinant exosporium-producing Bacillus cells that expresses a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide selected from the group consisting of an enzyme involved in theproduction or activation of a plant growth stimulating compound; anenzyme that degrades or modifies a bacterial, fungal, or plant nutrientsource; and a protein or peptide that protects a plant from a pathogen;and (ii) a targeting sequence that localizes the fusion protein to theexosporium of the Bacillus cereus family member; and b) at least onefurther and different particular biological control agent disclosedherein and/or a mutant of a specific strain of a microorganism disclosedherein having all identifying characteristics of the respective strain,and/or at least one metabolite produced by the respective strain thatexhibits activity against insects, mites, nematodes and/orphytopathogens in a synergistically effective amount.

A “synergistically effective amount” according to the present inventionrepresents a quantity of a combination of a recombinantexosporium-producing Bacillus cells that expresses a fusion protein andat least one further particular biological control agent describedherein that is more effective against insects, mites, nematodes and/orphytopathogens than the recombinant exosporium-producing Bacillus cellsthat expresses a fusion protein or such further biological control agentalone. A “synergistically effective amount” according to the presentinvention also represents a quantity of a combination of a recombinantexosporium-producing Bacillus cells that expresses a fusion protein andat least one further particular biological control agent describedherein that is more effective at enhancing plant growth and/or promotingplant health than the recombinant exosporium-producing Bacillus cellsthat expresses a fusion protein or such further biological control agentalone.

The present invention comprises each and every combination of each ofthe further particular biological control agents described herein withthe recombinant exosporium-producing Bacillus cells.

In a preferred embodiment the composition according to the presentinvention comprises at least one additional fungicide and/or at leastone insecticide, with the proviso that the recombinantexosporium-producing Bacillus cells, the insecticide and the fungicideare not identical.

The term “active compound” or “active ingredient” is used in the presentdescription to designate the recombinant exosporium-producing Bacilluscells, the at least one further biological control agent and/or a mutantof it having all identifying characteristics of the respective strain,and/or at least one metabolite produced by the respective strain thatexhibits activity against insects, mites, nematodes and/orphytopathogens, the at least one insecticide and the at least onefungicide.

Further Additives

One aspect of the present invention is to provide a composition asdescribed above additionally comprising at least one auxiliary selectedfrom the group consisting of extenders, solvents, spontaneity promoters,carriers, emulsifiers, dispersants, frost protectants, thickeners andadjuvants. Those compositions are referred to as formulations.

Accordingly, in one aspect of the present invention such formulations,and application forms prepared from them, are provided as cropprotection agents and/or pesticidal agents, such as drench, drip andspray liquors, comprising the composition of the invention. Theapplication forms may comprise further crop protection agents and/orpesticidal agents, and/or activity-enhancing adjuvants such aspenetrants, examples being vegetable oils such as, for example, rapeseedoil, sunflower oil, mineral oils such as, for example, liquid paraffins,alkyl esters of vegetable fatty acids, such as rapeseed oil or soybeanoil methyl esters, or alkanol alkoxylates, and/or spreaders such as, forexample, alkylsiloxanes and/or salts, examples being organic orinorganic ammonium or phosphonium salts, examples being ammoniumsulphate or diammonium hydrogen phosphate, and/or retention promoterssuch as dioctyl sulphosuccinate or hydroxypropylguar polymers and/orhumectants such as glycerol and/or fertilizers such as ammonium,potassium or phosphorous fertilizers, for example.

Examples of typical formulations include water-soluble liquids (SL),emulsifiable concentrates (EC), emulsions in water (EW), suspensionconcentrates (SC, SE, FS, OD), water-dispersible granules (WG), granules(GR) and capsule concentrates (CS); these and other possible types offormulation are described, for example, by Crop Life International andin Pesticide Specifications, Manual on Development and Use of FAO andWHO Specifications for Pesticides, FAO Plant Production and ProtectionPapers-173, prepared by the FAO/WHO Joint Meeting on PesticideSpecifications, 2004, ISBN: 9251048576. The formulations may compriseactive agrochemical compounds other than one or more active compounds ofthe invention.

The formulations or application forms in question preferably compriseauxiliaries, such as extenders, solvents, spontaneity promoters,carriers, emulsifiers, dispersants, frost protectants, biocides,thickeners and/or other auxiliaries, such as adjuvants, for example. Anadjuvant in this context is a component which enhances the biologicaleffect of the formulation, without the component itself having abiological effect. Examples of adjuvants are agents which promote theretention, spreading, attachment to the leaf surface, or penetration.

These formulations are produced in a known manner, for example by mixingthe active compounds with auxiliaries such as, for example, extenders,solvents and/or solid carriers and/or further auxiliaries, such as, forexample, surfactants. The formulations are prepared either in suitableplants or else before or during the application.

Suitable for use as auxiliaries are substances which are suitable forimparting to the formulation of the active compound or the applicationforms prepared from these formulations (such as, e.g., usable cropprotection agents, such as spray liquors or seed dressings) particularproperties such as certain physical, technical and/or biologicalproperties.

Suitable extenders are, for example, water, polar and nonpolar organicchemical liquids, for example from the classes of the aromatic andnon-aromatic hydrocarbons (such as paraffins, alkylbenzenes,alkylnaphthalenes, chlorobenzenes), the alcohols and polyols (which, ifappropriate, may also be substituted, etherified and/or esterified), theketones (such as acetone, cyclohexanone), esters (including fats andoils) and (poly)ethers, the unsubstituted and substituted amines,amides, lactams (such as N-alkylpyrrolidones) and lactones, thesulphones and sulphoxides (such as dimethyl sulphoxide).

If the extender used is water, it is also possible to employ, forexample, organic solvents as auxiliary solvents. Essentially, suitableliquid solvents are: aromatics such as xylene, toluene oralkylnaphthalenes, chlorinated aromatics and chlorinated aliphatichydrocarbons such as chlorobenzenes, chloroethylenes or methylenechloride, aliphatic hydrocarbons such as cyclohexane or paraffins, forexample petroleum fractions, mineral and vegetable oils, alcohols suchas butanol or glycol and also their ethers and esters, ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone,strongly polar solvents such as dimethylformamide and dimethylsulphoxide, and also water.

In principle it is possible to use all suitable solvents. Suitablesolvents are, for example, aromatic hydrocarbons, such as xylene,toluene or alkylnaphthalenes, for example, chlorinated aromatic oraliphatic hydrocarbons, such as chlorobenzene, chloroethylene ormethylene chloride, for example, aliphatic hydrocarbons, such ascyclohexane, for example, paraffins, petroleum fractions, mineral andvegetable oils, alcohols, such as methanol, ethanol, isopropanol,butanol or glycol, for example, and also their ethers and esters,ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone orcyclohexanone, for example, strongly polar solvents, such as dimethylsulphoxide, and water.

All suitable carriers may in principle be used. Suitable carriers are inparticular: for example, ammonium salts and ground natural minerals suchas kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite ordiatomaceous earth, and ground synthetic minerals, such as finelydivided silica, alumina and natural or synthetic silicates, resins,waxes and/or solid fertilizers. Mixtures of such carriers may likewisebe used. Carriers suitable for granules include the following: forexample, crushed and fractionated natural minerals such as calcite,marble, pumice, sepiolite, dolomite, and also synthetic granules ofinorganic and organic meals, and also granules of organic material suchas sawdust, paper, coconut shells, maize cobs and tobacco stalks.

Liquefied gaseous extenders or solvents may also be used. Particularlysuitable are those extenders or carriers which at standard temperatureand under standard pressure are gaseous, examples being aerosolpropellants, such as halogenated hydrocarbons, and also butane, propane,nitrogen and carbon dioxide.

Examples of emulsifiers and/or foam-formers, dispersants or wettingagents having ionic or nonionic properties, or mixtures of thesesurface-active substances, are salts of polyacrylic acid, salts oflignosulphonic acid, salts of phenolsulphonic acid ornaphthalenesulphonic acid, polycondensates of ethylene oxide with fattyalcohols or with fatty acids or with fatty amines, with substitutedphenols (preferably alkylphenols or arylphenols), salts ofsulphosuccinic esters, taurine derivatives (preferably alkyltaurates),phosphoric esters of polyethoxylated alcohols or phenols, fatty acidesters of polyols, and derivatives of the compounds containingsulphates, sulphonates and phosphates, examples being alkylarylpolyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates,protein hydrolysates, lignin-sulphite waste liquors and methylcellulose.The presence of a surface-active substance is advantageous if one of theactive compounds and/or one of the inert carriers is not soluble inwater and if application takes place in water.

Further auxiliaries that may be present in the formulations and in theapplication forms derived from them include colorants such as inorganicpigments, examples being iron oxide, titanium oxide, Prussian Blue, andorganic dyes, such as alizarin dyes, azo dyes and metal phthalocyaninedyes, and nutrients and trace nutrients, such as salts of iron,manganese, boron, copper, cobalt, molybdenum and zinc.

Stabilizers, such as low-temperature stabilizers, preservatives,antioxidants, light stabilizers or other agents which improve chemicaland/or physical stability may also be present. Additionally present maybe foam-formers or defoamers.

Furthermore, the formulations and application forms derived from themmay also comprise, as additional auxiliaries, stickers such ascarboxymethylcellulose, natural and synthetic polymers in powder,granule or latex form, such as gum arabic, polyvinyl alcohol, polyvinylacetate, and also natural phospholipids, such as cephalins andlecithins, and synthetic phospholipids. Further possible auxiliariesinclude mineral and vegetable oils.

There may possibly be further auxiliaries present in the formulationsand the application forms derived from them. Examples of such additivesinclude fragrances, protective colloids, binders, adhesives, thickeners,thixotropic substances, penetrants, retention promoters, stabilizers,sequestrants, complexing agents, humectants and spreaders. Generallyspeaking, the active compounds may be combined with any solid or liquidadditive commonly used for formulation purposes.

Suitable retention promoters include all those substances which reducethe dynamic surface tension, such as dioctyl sulphosuccinate, orincrease the viscoelasticity, such as hydroxypropylguar polymers, forexample.

Suitable penetrants in the present context include all those substanceswhich are typically used in order to enhance the penetration of activeagrochemical compounds into plants. Penetrants in this context aredefined in that, from the (generally aqueous) application liquor and/orfrom the spray coating, they are able to penetrate the cuticle of theplant and thereby increase the mobility of the active compounds in thecuticle. This property can be determined using the method described inthe literature (Baur, et al., 1997, Pesticide Science 51, 131-152).Examples include alcohol alkoxylates such as coconut fatty ethoxylate(10) or isotridecyl ethoxylate (12), fatty acid esters such as rapeseedor soybean oil methyl esters, fatty amine alkoxylates such astallowamine ethoxylate (15), or ammonium and/or phosphonium salts suchas ammonium sulphate or diammonium hydrogen phosphate, for example.

The formulations preferably comprise between 0.0001%and 98% by weight ofactive compound or, with particular preference, between 0.01% and 95% byweight of active compound, more preferably between 0.5% and 90% byweight of active compound, based on the weight of the formulation. Thecontent of the active compound is defined as the sum of the recombinantexosporium-producing Bacillus cells and the further particularbiological control agent described herein and/or a mutant of aparticular microorganism strain described herein having all identifyingcharacteristics of the respective strain, and/or at least one metaboliteproduced by the respective strain that exhibits activity againstinsects, mites, nematodes and/or phytopathogens, and fungicide and/orinsecticide, if present.

The active compound content of the application forms (crop protectionproducts) prepared from the formulations may vary within wide ranges.The active compound concentration of the application forms may besituated typically between 0.0001% and 95% by weight of active compound,preferably between 0.0001% and 1% by weight, based on the weight of theapplication form. Application takes place in a customary manner adaptedto the application forms.

Furthermore, in one aspect of the present invention a kit of parts isprovided comprising recombinant exosporium-producing Bacillus cells andat least one further particular biological control agent disclosedherein and/or a mutant of a specific strain of a microorganism disclosedherein having all identifying characteristics of the respective strain,and/or at least one metabolite produced by the respective strain thatexhibits activity against insects, mites, nematodes and/orphytopathogens in a synergistically effective amount in a spatiallyseparated arrangement.

In a further embodiment of the present invention the above-mentioned kitof parts further comprises at least one fungicide and/or at least oneinsecticide, with the proviso that the recombinant exosporium-producingBacillus cells, insecticide and fungicide are not identical. Thefungicide and/or the insecticide can be present either in therecombinant exosporium-producing Bacillus cereus family member-basedbiological control agent component of the kit of parts or in thecomponent of the kit of parts comprising the particular biologicalcontrol agent disclosed herein, being spatially separated or in both ofthese components. In one embodiment, the fungicide and the insecticideare present in the recombinant exosporium-producing Bacillus cereusfamily member-based biological control agent component.

Moreover, the kit of parts according to the present invention canadditionally comprise at least one auxiliary selected from the groupconsisting of extenders, solvents, spontaneity promoters, carriers,emulsifiers, dispersants, frost protectants, thickeners and adjuvants asmentioned below. This at least one auxiliary can be present either inthe recombinant exosporium-producing Bacillus cereus family member-basedbiological control agent component of the kit of parts or in thecomponent of the kit of parts comprising the particular biologicalcontrol agent disclosed herein, being spatially separated or in both ofthese components.

In another aspect of the present invention the composition as describedabove is used for reducing overall damage of plants and plant parts aswell as losses in harvested fruits or vegetables caused by insects,mites, nematodes and/or phytopathogens.

Furthermore, in another aspect of the present invention the compositionas described above increases the overall plant health.

The term “plant health” generally comprises various sorts ofimprovements of plants that are not connected to the control of pests.For example, advantageous properties that may be mentioned are improvedcrop characteristics including: emergence, crop yields, protein content,oil content, starch content, more developed root system, improved rootgrowth, improved root size maintenance, improved root effectiveness,improved stress tolerance (e.g., against drought, heat, salt, UV, water,cold), reduced ethylene (reduced production and/or inhibition ofreception), tittering increase, increase in plant height, bigger leafblade, less dead basal leaves, stronger tillers, greener leaf color,pigment content, photosynthetic activity, less input needed (such asfertilizers or water), less seeds needed, more productive tillers,earlier flowering, early grain maturity, less plant verse (lodging),increased shoot growth, enhanced plant vigor, increased plant stand andearly and better germination.

With regard to the use according to the present invention, improvedplant health preferably refers to improved plant characteristicsincluding: crop yield, more developed root system (improved rootgrowth), improved root size maintenance, improved root effectiveness,tittering increase, increase in plant height, bigger leaf blade, lessdead basal leaves, stronger tillers, greener leaf color, photosyntheticactivity, more productive tillers, enhanced plant vigor, and increasedplant stand.

With regard to the present invention, improved plant health preferablyespecially refers to improved plant properties selected from crop yield,more developed root system, improved root growth, improved root sizemaintenance, improved root effectiveness, tillering increase, andincrease in plant height.

The effect of a composition according to the present invention on planthealth as defined herein can be determined by comparing plants which aregrown under the same environmental conditions, whereby a part of saidplants is treated with a composition according to the present inventionand another part of said plants is not treated with a compositionaccording to the present invention. Instead, said other part is nottreated at all or is treated with a placebo (i.e., an applicationwithout a composition according to the invention such as an applicationwithout all active ingredients (i.e., without the recombinantexosporium-producing Bacillus cells as described herein and without afurther particular biological control agent as described herein), or anapplication without the recombinant exosporium-producing Bacillus cellsas described herein, or an application without a further particularbiological control agent disclosed herein.

The composition according to the present invention may be applied in anydesired manner, such as in the form of a seed coating, soil drench,and/or directly in-furrow and/or as a foliar spray and applied eitherpre-emergence, post-emergence or both. In other words, the compositioncan be applied to the seed, the plant or to harvested fruits andvegetables or to the soil wherein the plant is growing or wherein it isdesired to grow (plant's locus of growth).

Reducing the overall damage of plants and plant parts often results inhealthier plants and/or in an increase in plant vigor and yield.

Preferably, the composition according to the present invention is usedfor treating conventional or transgenic plants or seed thereof.

The present invention also relates to methods for stimulating plantgrowth using any of the compositions described above comprisingrecombinant exosporium-producing Bacillus cells that express a fusionprotein and at least one of the further particular biological controlagents described herein. The method for stimulating plant growthcomprises applying to a plant, a plant part, to the locus surroundingthe plant or in which the plant will be planted (e.g., soil or othergrowth medium) a composition comprising recombinant exosporium-producingBacillus cells that express a fusion protein comprising: (i) at leastone plant growth stimulating protein or peptide; and (ii) a targetingsequence, exosporium protein, or exosporium protein fragment, and atleast one further particular biological control agent disclosed hereinand/or a mutant of a specific strain of a microorganism disclosed hereinhaving all identifying characteristics of the respective strain, and/orat least one metabolite produced by the respective strain that exhibitsactivity against insects, mites, nematodes and/or phytopathogens in asynergistically effective amount.

In another aspect of the present invention a method for reducing overalldamage of plants and plant parts as well as losses in harvested fruitsor vegetables caused by insects, mites, nematodes and/or phytopathogensis provided comprising the step of simultaneously or sequentiallyapplying the recombinant exosporium-producing Bacillus cells and atleast one further particular biological control agent described hereinin a synergistically effective amount.

In one embodiment of the present method the composition furthercomprises at least one fungicide. In one aspect, the at least onefungicide is a synthetic fungicide. In a further aspect of thisembodiment, the at least one fungicide is selected from the followinggroup: bitertanol, bixafen, bromuconazole, carbendazim, carpropamid,dichlofluanid, fenamidone, fenhexamid, fentin acetate, fentin hydroxide,fluopicolide, fluopyram, fluoxastrobin, fluquinconazole, fosetyl,iprodione, iprovalicarb, isotianil, metominostrobin, ofurace,pencycuron, penflufen, prochloraz, propamocarb, propineb,prothioconazole, pyrimethanil, spiroxamine, tebuconazole, tolylfluanid,triadimefon, triadimenol, triazoxide, and trifloxystrobin.

In another embodiment, the composition comprises at least oneinsecticide in addition to the fungicide or in place of the fungicide,provided that the insecticide, the fungicide, the recombinantexosporium-producing Bacillus cells and the particular biologicalcontrol agent disclosed herein are not identical.

In one embodiment, the at least one insecticide is a syntheticinsecticide. In a further embodiment, the at least one insecticide isselected from the following group: acetamiprid, aldicarb, amitraz,beta-cyfluthrin, carbaryl, clothianidin, cyfluthrin, cypermethrin,deltamethrin, endosulfan, ethion, ethiprole, ethoprophos, fenamiphos,fenobucarb, fenthion, fipronil, flubendiamide, fluopyram,flupyradifurone, formetanate, heptanophos, imidacloprid, methamidophos,methiocarb, methomyl, niclosamide, oxydemeton-methyl, phosalone,silafluofen, spirodiclofen, spiromesifen, spirotetramat, thiacloprid,thiodicarb, tralomethrin, triazophos, triflumuron, and vamidothion.

The method of the present invention includes the following applicationmethods, namely both of the recombinant exosporium-producing Bacilluscells and the at least one further particular biological control agentdisclosed herein may be formulated into a single, stable compositionwith an agriculturally acceptable shelf life (so called“solo-formulation”), or being combined before or at the time of use (socalled “combined-formulations”).

If not mentioned otherwise, the expression “combination” stands for thevarious combinations of the recombinant exosporium-producing Bacilluscells and the at least one further particular biological control agentdisclosed herein, and optionally the at least one fungicide and/or theat least one insecticide, in a solo-formulation, in a single “ready-mix”form, in a combined spray mixture composed from solo-formulations, suchas a “tank-mix”, and especially in a combined use of the single activeingredients when applied in a sequential manner, i.e., one after theother within a reasonably short period, such as a few hours or days,e.g., 2 hours to 7 days. The order of applying the composition accordingto the present invention is not essential for working the presentinvention. Accordingly, the term “combination” also encompasses thepresence of the recombinant exosporium-producing Bacillus cells and theat least one further particular biological control agent disclosedherein, and optionally the at least one fungicide and/or insecticide onor in a plant to be treated or its surrounding, habitat or storagespace, e.g., after simultaneously or consecutively applying therecombinant exosporium-producing Bacillus cells and the at least onefurther particular biological control agent disclosed herein, andoptionally the at least one fungicide and/or the at least oneinsecticide to a plant its surrounding, habitat or storage space.

If the recombinant exosporium-producing Bacillus cells and the at leastone further particular biological control agent described herein, andoptionally the at least one fungicide and/or the at least oneinsecticide are employed or used in a sequential manner, it is preferredto treat the plants or plant parts (which includes seeds and plantsemerging from the seed), harvested fruits and vegetables according tothe following method: Firstly applying the at least one fungicide and/orthe at least one insecticide on the plant or plant parts, and secondlyapplying the further particular biological control agent describedherein and the recombinant exosporium-producing Bacillus cells to thesame plant or plant parts. By this application manner the amount ofresidues of insecticides/fungicides on the plant upon harvesting is aslow as possible. The time periods between the first and the secondapplication within a (crop) growing cycle may vary and depend on theeffect to be achieved. For example, the first application is done toprevent an infestation of the plant or plant parts with insects, mites,nematodes and/or phytopathogens (this is particularly the case whentreating seeds) or to combat the infestation with insects, mites,nematodes and/or phytopathogens (this is particularly the case whentreating plants and plant parts) and the second application is done toprevent or control the infestation with insects, mites, nematodes and/orphytopathogens and/or to promote plant growth. Control in this contextmeans that the composition comprising the recombinantexosporium-producing Bacillus cells and the particular biologicalcontrol agent disclosed herein are not able to fully exterminate thepests or phytopathogenic fungi but are able to keep the infestation onan acceptable level.

The present invention also provides methods of enhancing the killing,inhibiting, preventative and/or repelling activity of the compositionsof the present invention by multiple applications. In some otherembodiments, the compositions of the present invention are applied to aplant and/or plant part for two times, during any desired developmentstages or under any predetermined pest pressure, at an interval of about1 hour, about 5 hours, about 10 hours, about 24 hours, about two days,about 3 days, about 4 days, about 5 days, about 1 week, about 10 days,about two weeks, about three weeks, about 1 month or more. Still in someembodiments, the compositions of the present invention are applied to aplant and/or plant part for more than two times, for example, 3 times, 4times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more,during any desired development stages or under any predetermined pestpressure, at an interval of about 1 hour, about 5 hours, about 10 hours,about 24 hours, about two days, about 3 days, about 4 days, about 5days, about 1 week, about 10 days, about two weeks, about three weeks,about 1 month or more. The intervals between each application can varyif it is desired. One skilled in the art will be able to determine theapplication times and length of interval depending on plant species,plant pest species, and other factors.

By following the before mentioned steps, a very low level of residues ofthe at least one fungicide and/or at least one insecticide on thetreated plant, plant parts, and the harvested fruits and vegetables canbe achieved.

If not mentioned otherwise the treatment of plants or plant parts (whichincludes seeds and plants emerging from the seed), harvested fruits andvegetables with the composition according to the invention is carriedout directly or by action on their surroundings, habitat or storagespace using customary treatment methods, for example dipping, spraying,atomizing, irrigating, evaporating, dusting, fogging, broadcasting,foaming, painting, spreading-on, watering (drenching), drip irrigating.It is furthermore possible to apply the recombinant exosporium-producingBacillus cells, the at least one further particular biological controlagent described herein, and optionally the at least one fungicide and/orthe at least one insecticide as solo-formulation orcombined-formulations by the ultra-low volume method, or to inject thecomposition according to the present invention as a composition or assole-formulations into the soil (in-furrow).

The term “plant to be treated” encompasses every part of a plantincluding its root system and the material—e.g., soil or nutritionmedium—which is in a radius of at least 10 cm, 20 cm, 30 cm around thecaulis or bole of a plant to be treated or which is at least 10 cm, 20cm, 30 cm around the root system of said plant to be treated,respectively.

The amount of the recombinant exosporium-producing Bacillus cells, whichis used or employed in combination with at least one further particularbiological control agent described herein, optionally in the presence ofat least one fungicide and/or the at least one insecticide, depends onthe final formulation as well as size or type of the plant, plant parts,seeds, harvested fruits and vegetables to be treated. Usually, therecombinant exosporium-producing Bacillus cells to be employed or usedaccording to the invention is present in about 1% to about 80% (w/w),preferably in about 1% to about 60% (w/w), more preferably about 10% toabout 50% (w/w) of its solo-formulation or combined-formulation with theat least one further particular biological control agent describedherein, and optionally the fungicide and/or the at least oneinsecticide.

Also the amount of the at least one further particular biologicalcontrol agent disclosed herein which is used or employed in combinationwith the recombinant exosporium-producing Bacillus cells, optionally inthe presence of at least one fungicide and/or the at least oneinsecticide, depends on the final formulation as well as size or type ofthe plant, plant parts, seeds, harvested fruit or vegetable to betreated. Usually, the further particular biological control agentdescribed herein to be employed or used according to the invention ispresent in about 0.1% to about 80% (w/w), preferably 1% to about 60%(w/w), more preferably about 10% to about 50% (w/w) of itssolo-formulation or combined-formulation with the recombinantexosporium-producing Bacillus cells, and optionally the at least onefungicide and/or the at least one insecticide.

Application of the recombinant exosporium-producing Bacillus cells maybe effected as a foliar spray, as a soil treatment, and/or as a seedtreatment/dressing. When used as a foliar treatment, in one embodiment,about 1/16 to about 5 gallons of whole broth are applied per acre. Whenused as a soil treatment, in one embodiment, about 1 to about 5 gallonsof whole broth are applied per acre. When used for seed treatment about1/32 to about ¼ gallons of whole broth are applied per acre. For seedtreatment, the end-use formulation contains at least 1×10⁴, at least1×10⁵, at least 1×10⁶, 1×10⁷, at least 1×10⁸, at least 1×10⁹, at least1×10¹⁰ colony forming units per gram.

The recombinant exosporium-producing Bacillus cells and at least onefurther particular biological control agent disclosed herein, and ifpresent preferably also the fungicide and/or the insecticide are used oremployed in a synergistic weight ratio. The skilled person is able tofind out the synergistic weight ratios for the present invention byroutine methods. The skilled person understands that these ratios referto the ratio within a combined-formulation as well as to the calculativeratio of the recombinant exosporium-producing Bacillus cells describedherein and the at least one further particular biological control agentdisclosed herein when both components are applied as mono-formulationsto a plant to be treated. The skilled person can calculate this ratio bysimple mathematics since the volume and the amount of the recombinantexosporium-producing Bacillus cells and the at least one furtherparticular biological control agent disclosed herein, respectively, in amono-formulation is known to the skilled person.

The ratio can be calculated based on the amount of the at least onefurther particular biological control agent disclosed herein, at thetime point of applying said component of a combination according to theinvention to a plant or plant part and the amount of the recombinantexosporium-producing Bacillus cells shortly prior (e.g., 48 h, 24 h, 12h, 6 h, 2 h, 1 h) or at the time point of applying said component of acombination according to the invention to a plant or plant part.

The application of the recombinant exosporium-producing Bacillus cellsand the at least one further particular biological control agentdisclosed herein to a plant or a plant part can take placesimultaneously or at different times as long as both components arepresent on or in the plant after the application(s). In cases where therecombinant exosporium-producing Bacillus cells and further particularbiological control agent disclosed herein are applied at different timesand the further particular biological control agent disclosed herein isapplied prior to the recombinant exosporium-producing Bacillus cells,the skilled person can determine the concentration of further particularbiological control agent disclosed herein on/in a plant by chemicalanalysis known in the art, at the time point or shortly before the timepoint of applying the recombinant exosporium-producing Bacillus cells.Vice versa, when the recombinant exosporium-producing Bacillus cells isapplied to a plant first, the concentration of the recombinantexosporium-producing Bacillus cells can be determined using tests whichare also known in the art, at the time point or shortly before the timepoint of applying the further particular biological control agentdisclosed herein.

In particular, in one embodiment the synergistic weight ratio of therecombinant exosporium-producing Bacillus cells (i.e., the unformulatedspore preparation) and the at least one further particular biologicalcontrol agent disclosed herein lies in the range of 1:1000 to 1000:1; inthe range of 1:500 to 500:1; in the range of 1:300 to 500:1. Additionalratios are between 20:1 and 1:20, such as 10:1, 5:1 or 2:1. Inembodiments in which the biological control agent is Bacillus-based theweight to weight ratio should be applied to the unformulated Bacillusspore preparation. In one aspect of this embodiment, the sporepreparations of both the recombinant exosporium-producing Bacillus cellsand the Bacillus-based biological control agent is dried sporepreparation containing at least about 1×10⁴ cfu/g, at least about 1×10⁵cfu/g, at least about 1×10⁶ cfu/g at least about 1×10⁷ cfu/g, at leastabout 1×10⁸ cfu/g, at least about 1×10⁹ cfu/g, at least about 1×10¹⁰cfu/g, and at least about 1×10¹¹ cfu/g. In another embodiment the colonyforming unit to colony forming unit ratio of recombinantexosporium-producing Bacillus cells and the Bacillus-based particularbiological control agent described herein lies in the range of 1:100,000to 100,000 to 1, in the range of 1:10,000 to 10,000:1, in the range of1:1000 to 1000:1, in the range of 1:500 to 500:1, in the range of 1:100to 100:1, in the range of 1:10 to 10:1, in the range of 1:5 to 5:1, andin the range of 1:1.

In one embodiment of the present invention, the concentration of therecombinant exosporium-producing Bacillus cereus family member-basedbiological control agent after dispersal is at least 50 g/ha, such as50-7500 g/ha, 50-2500 g/ha, 50-1500 g/ha; at least 250 g/ha (hectare),at least 500 g/ha or at least 800 g/ha.

The application rate of composition to be employed or used according tothe present invention may vary. The skilled person is able to find theappropriate application rate by way of routine experiments.

In another aspect of the present invention a seed treated with thecomposition as described above is provided.

The control of insects, mites, nematodes and/or phytopathogens bytreating the seed of plants has been known for a long time and is asubject of continual improvements. Nevertheless, the treatment of seedentails a series of problems which cannot always be solved in asatisfactory manner. Thus, it is desirable to develop methods forprotecting the seed and the germinating plant that remove the need for,or at least significantly reduce, the additional delivery of cropprotection compositions in the course of storage, after sowing or afterthe emergence of the plants. It is desirable, furthermore, to optimizethe amount of active ingredient employed in such a way as to provide thebest-possible protection to the seed and the germinating plant fromattack by insects, mites, nematodes and/or phytopathogens, but withoutcausing damage to the plant itself by the active ingredient employed. Inparticular, methods for treating seed ought also to take intoconsideration the intrinsic insecticidal and/or nematicidal propertiesof pest-resistant or pest-tolerant transgenic plants, in order toachieve optimum protection of the seed and of the germinating plant witha minimal use of crop protection compositions.

The present invention therefore also relates in particular to a methodfor protecting seed and germinating plants from attack by pests, bytreating the seed with the recombinant exosporium-producing Bacilluscells as defined above and at least one further biological control agentselected from particular microorganisms disclosed herein and/or a mutantof a specific strain of microorganism disclosed herein having allidentifying characteristics of the respective strain, and/or at leastone metabolite produced by the respective strain that exhibits activityagainst insects, mites, nematodes and/or phytopathogens and optionallyat least one fungicide and/or optionally at least one insecticide of theinvention. The method of the invention for protecting seed andgerminating plants from attack by pests encompasses a method in whichthe seed is treated simultaneously in one operation with the recombinantexosporium-producing Bacillus cells and the at least one furtherparticular biological control agent described herein, and optionally theat least one fungicide and/or the at least one insecticide. It alsoencompasses a method in which the seed is treated at different timeswith the recombinant exosporium-producing Bacillus cells and the atleast one further particular biological control agent disclosed herein,and optionally the at least one fungicide and/or the at least oneinsecticide.

The invention likewise relates to the use of the composition of theinvention for treating seed for the purpose of protecting the seed andthe resultant plant against insects, mites, nematodes and/orphytopathogens.

The invention also relates to seed which at the same time has beentreated with a the recombinant exosporium-producing Bacillus cells andat least one further particular biological control agent describedherein, and optionally at least one fungicide and/or the at least oneinsecticide. The invention further relates to seed which has beentreated at different times with the recombinant exosporium-producingBacillus cells and the at least one further particular biologicalcontrol agent disclosed herein and optionally the at least one fungicideand/or the at least one insecticide. In the case of seed which has beentreated at different times with the recombinant exosporium-producingBacillus cells and the at least one further particular biologicalcontrol agent disclosed herein, and optionally the at least onefungicide and/or the at least one insecticide, the individual activeingredients in the composition of the invention may be present indifferent layers on the seed.

Furthermore, the invention relates to seed which, following treatmentwith the composition of the invention, is subjected to a film-coatingprocess in order to prevent dust abrasion of the seed.

One of the advantages of the present invention is that, owing to theparticular systemic properties of the compositions of the invention, thetreatment of the seed with these compositions provides protection frominsects, mites, nematodes and/or phytopathogens not only to the seeditself but also to the plants originating from the seed, after they haveemerged. In this way, it may not be necessary to treat the crop directlyat the time of sowing or shortly thereafter.

A further advantage is to be seen in the fact that, through thetreatment of the seed with composition of the invention, germination andemergence of the treated seed may be promoted.

It is likewise considered to be advantageous composition of theinvention may also be used, in particular, on transgenic seed.

It is also stated that the composition of the invention may be used incombination with agents of the signalling technology, as a result ofwhich, for example, colonization with symbionts is improved, such asrhizobia, mycorrhiza and/or endophytic bacteria, for example, isenhanced, and/or nitrogen fixation is optimized.

The compositions of the invention are suitable for protecting seed ofany variety of plant which is used in agriculture, in greenhouses, inforestry or in horticulture. More particularly, the seed in question isthat of cereals (e.g., wheat, barley, rye, oats and millet), maize,cotton, soybeans, rice, potatoes, sunflower, coffee, tobacco, canola,oilseed rape, beets (e.g., sugar beet and fodder beet), peanuts,vegetables (e.g., tomato, cucumber, bean, brassicas, onions andlettuce), fruit plants, lawns and ornamentals. Particularly important isthe treatment of the seed of cereals (such as wheat, barley, rye andoats) maize, soybeans, cotton, canola, oilseed rape and rice.

As already mentioned above, the treatment of transgenic seed with thecomposition of the invention is particularly important. The seed inquestion here is that of plants which generally contain at least oneheterologous gene that controls the expression of a polypeptide having,in particular, insecticidal and/or nematicidal properties. Theseheterologous genes in transgenic seed may come from microorganisms suchas Bacillus, Rhizobium, Pseudomonas, Serratia, Trichoderma, Clavibacter,Glomus or Gliocladium. The present invention is particularly suitablefor the treatment of transgenic seed which contains at least oneheterologous gene from Bacillus sp. With particular preference, theheterologous gene in question comes from Bacillus thuringiensis.

For the purposes of the present invention, the composition of theinvention is applied alone or in a suitable formulation to the seed. Theseed is preferably treated in a condition in which its stability is suchthat no damage occurs in the course of the treatment. Generallyspeaking, the seed may be treated at any point in time betweenharvesting and sowing. Typically, seed is used which has been separatedfrom the plant and has had cobs, hulls, stems, husks, hair or pulpremoved. Thus, for example, seed may be used that has been harvested,cleaned and dried to a moisture content of less than 15% by weight.Alternatively, seed can also be used that after drying has been treatedwith water, for example, and then dried again.

When treating seed it is necessary, generally speaking, to ensure thatthe amount of the composition of the invention, and/or of otheradditives, that is applied to the seed is selected such that thegermination of the seed is not adversely affected, and/or that the plantwhich emerges from the seed is not damaged. This is the case inparticular with active ingredients which may exhibit phytotoxic effectsat certain application rates.

The compositions of the invention can be applied directly, in otherwords without comprising further components and without having beendiluted. As a general rule, it is preferable to apply the compositionsin the form of a suitable formulation to the seed. Suitable formulationsand methods for seed treatment are known to the skilled person and aredescribed in, for example, the following documents: U.S. Pat. Nos.4,272,417 A; 4,245,432 A; 4,808,430 A; 5,876,739 A; U.S. PatentPublication No. 2003/0176428 A1; WO 2002/080675 A1; WO 2002/028186 A2.

The combinations which can be used in accordance with the invention maybe converted into the customary seed-dressing formulations, such assolutions, emulsions, suspensions, powders, foams, slurries or othercoating compositions for seed, and also ULV formulations.

These formulations are prepared in a known manner, by mixing compositionwith customary adjuvants, such as, for example, customary extenders andalso solvents or diluents, colorants, wetters, dispersants, emulsifiers,antifoams, preservatives, secondary thickeners, stickers, gibberellins,and also water.

Colorants which may be present in the seed-dressing formulations whichcan be used in accordance with the invention include all colorants whichare customary for such purposes. In this context it is possible to usenot only pigments, which are of low solubility in water, but alsowater-soluble dyes. Examples include the colorants known under thedesignations Rhodamin B, C.I. Pigment Red 112 and C.I. Solvent Red 1.

Wetters which may be present in the seed-dressing formulations which canbe used in accordance with the invention include all of the substanceswhich promote wetting and which are customary in the formulation ofactive agrochemical ingredients. Use may be made preferably ofalkylnaphthalenesulphonates, such as diisopropyl- ordiisobutyl-naphthalenesulphonates.

Dispersants and/or emulsifiers which may be present in the seed-dressingformulations which can be used in accordance with the invention includeall of the nonionic, anionic and cationic dispersants that are customaryin the formulation of active agrochemical ingredients. Use may be madepreferably of nonionic or anionic dispersants or of mixtures of nonionicor anionic dispersants. Suitable nonionic dispersants are, inparticular, ethylene oxide-propylene oxide block polymers, alkylphenolpolyglycol ethers and also tristryrylphenol polyglycol ethers, and thephosphated or sulphated derivatives of these. Suitable anionicdispersants are, in particular, lignosulphonates, salts of polyacrylicacid, and arylsulphonate-formaldehyde condensates.

Antifoams which may be present in the seed-dressing formulations whichcan be used in accordance with the invention include all of the foaminhibitors that are customary in the formulation of active agrochemicalingredients. Use may be made preferably of silicone antifoams andmagnesium stearate.

Preservatives which may be present in the seed-dressing formulationswhich can be used in accordance with the invention include all of thesubstances which can be employed for such purposes in agrochemicalcompositions. Examples include dichlorophen and benzyl alcoholhemiformal.

Secondary thickeners which may be present in the seed-dressingformulations which can be used in accordance with the invention includeall substances which can be used for such purposes in agrochemicalcompositions. Those contemplated with preference include cellulosederivatives, acrylic acid derivatives, xanthan, modified clays andhighly disperse silica.

Stickers which may be present in the seed-dressing formulations whichcan be used in accordance with the invention include all customarybinders which can be used in seed-dressing products. Preferred mentionmay be made of polyvinylpyrrolidone, polyvinyl acetate, polyvinylalcohol and tylose.

Gibberellins which may be present in the seed-dressing formulationswhich can be used in accordance with the invention include preferablythe gibberellins A1, A3 (=gibberellic acid), A4 and A7, with gibberellicacid being used with particular preference. The gibberellins are known(cf. R. Wegler, “Chemie der Pflanzenschutz—andSchädlingsbekämpfungsmittel”, Volume 2, Springer Verlag, 1970, pp.401-412).

The seed-dressing formulations which can be used in accordance with theinvention may be used, either directly or after prior dilution withwater, to treat seed of any of a wide variety of types. Accordingly, theconcentrates or the preparations obtainable from them by dilution withwater may be employed to dress the seed of cereals, such as wheat,barley, rye, oats and triticale, and also the seed of maize, rice,oilseed rape, peas, beans, cotton, sunflowers and beets, or else theseed of any of a very wide variety of vegetables. The seed-dressingformulations which can be used in accordance with the invention, ortheir diluted preparations, may also be used to dress seed of transgenicplants. In that case, additional synergistic effects may occur ininteraction with the substances formed through expression.

For the treatment of seed with the seed-dressing formulations which canbe used in accordance with the invention, or with the preparationsproduced from them by addition of water, suitable mixing equipmentincludes all such equipment which can typically be employed for seeddressing. More particularly, the procedure when carrying out seeddressing is to place the seed in a mixer, to add the particular desiredamount of seed-dressing formulations, either as such or followingdilution with water beforehand, and to carry out mixing until thedistribution of the formulation on the seed is uniform. This may befollowed by a drying operation.

The application rate of the seed-dressing formulations which can be usedin accordance with the invention may be varied within a relatively widerange. It is guided by the particular amount of the recombinantexosporium-producing Bacillus cells and the at least one furtherparticular biological control agent described herein in theformulations, and by the seed. The application rates in the case of thecomposition are situated generally at between 0.001 and 50 g perkilogram of seed, preferably between 0.01 and 15 g per kilogram of seed.

Furthermore, the composition according to the present inventionpreferably has potent microbicidal activity and can be used for controlof unwanted microorganisms, such as fungi and bacteria, in cropprotection and in the protection of materials.

The invention also relates to a method for controlling unwantedmicroorganisms, characterized in that the inventive composition isapplied to the phytopathogenic fungi, phytopathogenic bacteria and/ortheir habitat.

Fungicides can be used in crop protection for control of phytopathogenicfungi. They are characterized by an outstanding efficacy against a broadspectrum of phytopathogenic fungi, including soilborne pathogens, whichare in particular members of the classes Plasmodiophoromycetes,Peronosporomycetes (Syn. Oomycetes), Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes and Deuteromycetes (Syn. Fungi imperfecti).Some fungicides are systemically active and can be used in plantprotection as foliar, seed dressing or soil fungicide. Furthermore, theyare suitable for combating fungi, which inter alia infest wood or rootsof plant.

Bactericides can be used in crop protection for control ofPseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceaeand Streptomycetaceae.

Non-limiting examples of pathogens of fungal diseases which can betreated in accordance with the invention include:

diseases caused by powdery mildew pathogens, for example Blumeriaspecies, for example Blumeria graminis; Podosphaera species, for examplePodosphaera leucotricha; Sphaerotheca species, for example Sphaerothecafuliginea; Uncinula species, for example Uncinula necator;

diseases caused by rust disease pathogens, for example Gymnosporangiumspecies, for example Gymnosporangium sabinae; Hemileia species, forexample Hemileia vastatrix; Phakopsora species, for example Phakopsorapachyrhizi and Phakopsora meibomiae; Puccinia species, for examplePuccinia recondite, P. triticina, P. graminis or P. striiformis or P.hordei; Uromyces species, for example Uromyces appendiculatus;

diseases caused by pathogens from the group of the Oomycetes, forexample Albugo species, for example Algubo candida; Bremia species, forexample Bremia lactucae; Peronospora species, for example Peronosporapisi, P. parasitica or P. brassicae; Phytophthora species, for examplePhytophthora infestans; Plasmopara species, for example Plasmoparaviticola; Pseudoperonospora species, for example Pseudoperonosporahumuli or Pseudoperonospora cubensis; Pythium species, for examplePythium ultimum;

leaf blotch diseases and leaf wilt diseases caused, for example, byAlternaria species, for example Alternaria solani; Cercospora species,for example Cercospora beticola; Cladiosporium species, for exampleCladiosporium cucumerinum; Cochliobolus species, for exampleCochliobolus sativus (conidia form: Drechslera, Syn: Helminthosporium),Cochliobolus miyabeanus; Colletotrichum species, for exampleColletotrichum lindemuthanium; Cycloconium species, for exampleCycloconium oleaginum; Diaporthe species, for example Diaporthe citri;Elsinoe species, for example Elsinoe fawcettii; Gloeosporium species,for example Gloeosporium laeticolor; Glomerella species, for exampleGlomerella cingulata; Guignardia species, for example Guignardiabidwelli; Leptosphaeria species, for example Leptosphaeria maculans,Leptosphaeria nodorum; Magnaporthe species, for example Magnaporthegrisea; Microdochium species, for example Microdochium nivale;Mycosphaerella species, for example Mycosphaerella graminicola, M.arachidicola and M. fijiensis; Phaeosphaeria species, for examplePhaeosphaeria nodorum; Pyrenophora species, for example Pyrenophorateres, Pyrenophora tritici repentis; Ramularia species, for exampleRamularia collo-cygni, Ramularia areola; Rhynchosporium species, forexample Rhynchosporium secalis; Septoria species, for example Septoriaapii, Septoria lycopersii; Typhula species, for example Typhulaincarnata; Venturia species, for example Venturia inaequalis;

root and stem diseases caused, for example, by Corticium species, forexample Corticium graminearum; Fusarium species, for example Fusariumoxysporum; Gaeumannomyces species, for example Gaeumannomyces graminis;Rhizoctonia species, such as, for example Rhizoctonia solani;Sarocladium diseases caused for example by Sarocladium oryzae;Sclerotium diseases caused for example by Sclerotium oryzae; Tapesiaspecies, for example Tapesia acuformis; Thielaviopsis species, forexample Thielaviopsis basicola;

ear and panicle diseases (including corn cobs) caused, for example, byAlternaria species, for example Alternaria spp.; Aspergillus species,for example Aspergillus flavus; Cladosporium species, for exampleCladosporium cladosporioides; Claviceps species, for example Clavicepspurpurea; Fusarium species, for example Fusarium culmorum; Gibberellaspecies, for example Gibberella zeae; Monographella species, for exampleMonographella nivalis; Septoria species, for example Septoria nodorum;

diseases caused by smut fungi, for example Sphacelotheca species, forexample Sphacelotheca reiliana; Tilletia species, for example Tilletiacaries, T. controversa; Urocystis species, for example Urocystisocculta; Ustilago species, for example Ustilago nuda, U. nuda tritici;

fruit rot caused, for example, by Aspergillus species, for exampleAspergillus flavus; Botrytis species, for example Botrytis cinerea;Penicillium species, for example Penicillium expansum and P.purpurogenum; Sclerotinia species, for example Sclerotinia sclerotiorum;Verticilium species, for example Verticilium alboatrum;

seed and soilborne decay, mould, wilt, rot and damping-off diseasescaused, for example, by Alternaria species, caused for example byAlternaria brassicicola; Aphanomyces species, caused for example byAphanomyces euteiches; Ascochyta species, caused for example byAscochyta lentis; Aspergillus species, caused for example by Aspergillusflavus; Cladosporium species, caused for example by Cladosporiumherbarum; Cochliobolus species, caused for example by Cochliobolussativus; (Conidiaform: Drechslera, Bipolaris Syn: Helminthosporium);Colletotrichum species, caused for example by Colletotrichum coccodes;Fusarium species, caused for example by Fusarium culmorum; Gibberellaspecies, caused for example by Gibberella zeae; Macrophomina species,caused for example by Macrophomina phaseolina; Monographella species,caused for example by Monographella nivalis; Penicillium species, causedfor example by Penicillium expansum; Phoma species, caused for exampleby Phoma lingam; Phomopsis species, caused for example by Phomopsissojae; Phytophthora species, caused for example by Phytophthoracactorum; Pyrenophora species, caused for example by Pyrenophoragraminea; Pyricularia species, caused for example by Pyricularia oryzae;Pythium species, caused for example by Pythium ultimum; Rhizoctoniaspecies, caused for example by Rhizoctonia solani; Rhizopus species,caused for example by Rhizopus oryzae; Sclerotium species, caused forexample by Sclerotium rolfsii; Septoria species, caused for example bySeptoria nodorum; Typhula species, caused for example by Typhulaincarnata; Verticillium species, caused for example by Verticilliumdahliae;

cancers, galls and witches' broom caused, for example, by Nectriaspecies, for example Nectria galligena;

wilt diseases caused, for example, by Monilinia species, for exampleMonilinia laxa;

leaf blister or leaf curl diseases caused, for example, by Exobasidiumspecies, for example Exobasidium vexans;

Taphrina species, for example Taphrina deformans;

decline diseases of wooden plants caused, for example, by Esca disease,caused for example by Phaemoniella clamydospora, Phaeoacremoniumaleophilum and Fomitiporia mediterranea; Eutypa dyeback, caused forexample by Eutypa lata; Ganoderma diseases caused for example byGanoderma boninense; Rigidoporus diseases caused for example byRigidoporus lignosus;

diseases of flowers and seeds caused, for example, by Botrytis species,for example Botrytis cinerea;

diseases of plant tubers caused, for example, by Rhizoctonia species,for example Rhizoctonia solani; Helminthosporium species, for exampleHelminthosporium solani;

Club root caused, for example, by Plasmodiophora species, for examplePlamodiophora brassicae;

diseases caused by bacterial pathogens, for example Xanthomonas species,for example Xanthomonas campestris pv. oryzae; Pseudomonas species, forexample Pseudomonas syringae pv. lachrymans; Erwinia species, forexample Erwinia amylovora.

The following diseases of soya beans can be controlled with preference:

Fungal diseases on leaves, stems, pods and seeds caused, for example, byAlternaria leaf spot (Alternaria spec. atrans tenuissima), Anthracnose(Colletotrichum gloeosporoides dematium var. truncatum), brown spot(Septoria glycines), cercospora leaf spot and blight (Cercosporakikuchii), choanephora leaf blight (Choanephora infundibulifera trispora(Syn.)), dactuliophora leaf spot (Dactuliophora glycines), downy mildew(Peronospora manshurica), drechslera blight (Drechslera glycini),frogeye leaf spot (Cercospora sojina), leptosphaerulina leaf spot(Leptosphaerulina trifolii), phyllostica leaf spot (Phyllostictasojaecola), pod and stem blight (Phomopsis sojae), powdery mildew(Microsphaera diffusa), pyrenochaeta leaf spot (Pyrenochaeta glycines),rhizoctonia aerial, foliage, and web blight (Rhizoctonia solani), rust(Phakopsora pachyrhizi, Phakopsora meibomiae), scab (Sphacelomaglycines), stemphylium leaf blight (Stemphylium botryosum), target spot(Corynespora cassiicola).

Fungal diseases on roots and the stem base caused, for example, by blackroot rot (Calonectria crotalariae), charcoal rot (Macrophominaphaseolina), fusarium blight or wilt, root rot, and pod and collar rot(Fusarium oxysporum, Fusarium orthoceras, Fusarium semitectum, Fusariumequiseti), mycoleptodiscus root rot (Mycoleptodiscus terrestris),neocosmospora (Neocosmospora vasinfecta), pod and stem blight (Diaporthephaseolorum), stem canker (Diaporthe phaseolorum var. caulivora),phytophthora rot (Phytophthora megasperma), brown stem rot (Phialophoragregata), pythium rot (Pythium aphanidermatum, Pythium irregulare,Pythium debaryanum, Pythium myriotylum, Pythium ultimum), rhizoctoniaroot rot, stem decay, and damping-off (Rhizoctonia solani), sclerotiniastem decay (Sclerotinia sclerotiorum), sclerotinia southern blight(Sclerotinia rolfsii), thielaviopsis root rot (Thielaviopsis basicola).

The inventive compositions can be used for curative orprotective/preventive control of phytopathogenic fungi. The inventiontherefore also relates to curative and protective methods forcontrolling phytopathogenic fungi by the use of the inventivecomposition, which is applied to the seed, the plant or plant parts, thefruit or the soil in which the plants grow.

The fact that the composition is well tolerated by plants at theconcentrations required for controlling plant diseases allows thetreatment of above-ground parts of plants, of propagation stock andseeds, and of the soil.

According to the invention all plants and plant parts can be treated. Byplants is meant all plants and plant populations such as desirable andundesirable wild plants, cultivars and plant varieties (whether or notprotectable by plant variety or plant breeder's rights). Cultivars andplant varieties can be plants obtained by conventional propagation andbreeding methods which can be assisted or supplemented by one or morebiotechnological methods such as by use of double haploids, protoplastfusion, random and directed mutagenesis, molecular or genetic markers orby bioengineering and genetic engineering methods. By plant parts ismeant all above ground and below ground parts and organs of plants suchas shoot, leaf, blossom and root, whereby for example leaves, needles,stems, branches, blossoms, fruiting bodies, fruits and seed as well asroots, corms and rhizomes are listed. Crops and vegetative andgenerative propagating material, for example cuttings, corms, rhizomes,runners and seeds also belong to plant parts.

The inventive composition, when it is well tolerated by plants, hasfavourable homeotherm toxicity and is well tolerated by the environment,is suitable for protecting plants and plant organs, for enhancingharvest yields, for improving the quality of the harvested material. Itcan preferably be used as crop protection composition. It is activeagainst normally sensitive and resistant species and against all or somestages of development.

Plants which can be treated in accordance with the invention include thefollowing main crop plants: maize, soya bean, alfalfa, cotton,sunflower, Brassica oil seeds such as Brassica napus (e.g., canola,rapeseed), Brassica rapa, B. juncea (e.g., (field) mustard) and Brassicacarinata, Arecaceae sp. (e.g., oilpalm, coconut), rice, wheat, sugarbeet, sugar cane, oats, rye, barley, millet and sorghum, triticale,flax, nuts, grapes and vine and various fruit and vegetables fromvarious botanic taxa, e.g., Rosaceae sp. (e.g., pome fruits such asapples and pears, but also stone fruits such as apricots, cherries,almonds, plums and peaches, and berry fruits such as strawberries,raspberries, red and black currant and gooseberry), Ribesioidae sp.,Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp.,Moraceae sp., Oleaceae sp. (e.g., olive tree), Actinidaceae sp.,Lauraceae sp. (e.g., avocado, cinnamon, camphor), Musaceae sp. (e.g.,banana trees and plantations), Rubiaceae sp. (e.g., coffee), Theaceaesp. (e.g., tea), Sterculiceae sp., Rutaceae sp. (e.g., lemons, oranges,mandarins and grapefruit); Solanaceae sp. (e.g., tomatoes, potatoes,peppers, capsicum, aubergines, tobacco), Liliaceae sp., Compositae sp.(e.g., lettuce, artichokes and chicory—including root chicory, endive orcommon chicory), Umbelliferae sp. (e.g., carrots, parsley, celery andceleriac), Cucurbitaceae sp. (e.g., cucumbers—including gherkins,pumpkins, watermelons, calabashes and melons), Alliaceae sp. (e.g.,leeks and onions), Cruciferae sp. (e.g., white cabbage, red cabbage,broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes,horseradish, cress and chinese cabbage), Leguminosae sp. (e.g., peanuts,peas, lentils and beans—e.g., common beans and broad beans),Chenopodiaceae sp. (e.g., Swiss chard, fodder beet, spinach, beetroot),Linaceae sp. (e.g., hemp), Cannabeacea sp. (e.g., cannabis), Malvaceaesp. (e.g., okra, cocoa), Papaveraceae (e.g., poppy), Asparagaceae (e.g.,asparagus); useful plants and ornamental plants in the garden and woodsincluding turf, lawn, grass and Stevia rebaudiana; and in each casegenetically modified types of these plants.

Depending on the plant species or plant cultivars, their location andgrowth conditions (soils, climate, vegetation period, diet), using oremploying the composition according to the present invention thetreatment according to the invention may also result in super-additive(“synergistic”) effects. Thus, for example, by using or employinginventive composition in the treatment according to the invention,reduced application rates and/or a widening of the activity spectrumand/or an increase in the activity better plant growth, increasedtolerance to high or low temperatures, increased tolerance to drought orto water or soil salt content, increased flowering performance, easierharvesting, accelerated maturation, higher harvest yields, biggerfruits, larger plant height, greener leaf color, earlier flowering,higher quality and/or a higher nutritional value of the harvestedproducts, higher sugar concentration within the fruits, better storagestability and/or processability of the harvested products are possible,which exceed the effects which were actually to be expected.

At certain application rates of the inventive composition in thetreatment according to the invention may also have a strengtheningeffect in plants. The defense system of the plant against attack byunwanted phytopathogenic fungi and/or microorganisms and/or viruses ismobilized. Plant-strengthening (resistance-inducing) substances are tobe understood as meaning, in the present context, those substances orcombinations of substances which are capable of stimulating the defensesystem of plants in such a way that, when subsequently inoculated withunwanted phytopathogenic fungi and/or microorganisms and/or viruses, thetreated plants display a substantial degree of resistance to thesephytopathogenic fungi and/or microorganisms and/or viruses. Thus, byusing or employing composition according to the present invention in thetreatment according to the invention, plants can be protected againstattack by the abovementioned pathogens within a certain period of timeafter the treatment. The period of time within which protection iseffected generally extends from 1 to 10 days, preferably 1 to 7 days,after the treatment of the plants with the active compounds.

Plants and plant cultivars which are also preferably to be treatedaccording to the invention are resistant against one or more bioticstresses, i.e., said plants show a better defense against animal andmicrobial pests, such as against nematodes, insects, mites,phytopathogenic fungi, bacteria, viruses and/or viroids.

Plants and plant cultivars which may also be treated according to theinvention are those plants which are resistant to one or more abioticstresses, i.e., that already exhibit an increased plant health withrespect to stress tolerance. Abiotic stress conditions may include, forexample, drought, cold temperature exposure, heat exposure, osmoticstress, flooding, increased soil salinity, increased mineral exposure,ozon exposure, high light exposure, limited availability of nitrogennutrients, limited availability of phosphorus nutrients, shadeavoidance. Preferably, the treatment of these plants and cultivars withthe composition of the present invention additionally increases theoverall plant health (cf. above).

Plants and plant cultivars which may also be treated according to theinvention, are those plants characterized by enhanced yieldcharacteristics, i.e., that already exhibit an increased plant healthwith respect to this feature. Increased yield in said plants can be theresult of, for example, improved plant physiology, growth anddevelopment, such as water use efficiency, water retention efficiency,improved nitrogen use, enhanced carbon assimilation, improvedphotosynthesis, increased germination efficiency and acceleratedmaturation.

Yield can furthermore be affected by improved plant architecture (understress and non-stress conditions), including but not limited to, earlyflowering, flowering control for hybrid seed production, seedling vigor,plant size, internode number and distance, root growth, seed size, fruitsize, pod size, pod or ear number, seed number per pod or ear, seedmass, enhanced seed filling, reduced seed dispersal, reduced poddehiscence and lodging resistance. Further yield traits include seedcomposition, such as carbohydrate content, protein content, oil contentand composition, nutritional value, reduction in anti-nutritionalcompounds, improved processability and better storage stability.Preferably, the treatment of these plants and cultivars with thecomposition of the present invention additionally increases the overallplant health (cf. above).

Plants that may be treated according to the invention are hybrid plantsthat already express the characteristic of heterosis or hybrid vigorwhich results in generally higher yield, vigor, health and resistancetowards biotic and abiotic stress factors. Such plants are typicallymade by crossing an inbred male-sterile parent line (the female parent)with another inbred male-fertile parent line (the male parent). Hybridseed is typically harvested from the male sterile plants and sold togrowers. Male sterile plants can sometimes (e.g., in corn) be producedby detasseling, i.e., the mechanical removal of the male reproductiveorgans (or males flowers) but, more typically, male sterility is theresult of genetic determinants in the plant genome. In that case, andespecially when seed is the desired product to be harvested from thehybrid plants it is typically useful to ensure that male fertility inthe hybrid plants is fully restored. This can be accomplished byensuring that the male parents have appropriate fertility restorer geneswhich are capable of restoring the male fertility in hybrid plants thatcontain the genetic determinants responsible for male-sterility. Geneticdeterminants for male sterility may be located in the cytoplasm.Examples of cytoplasmic male sterility (CMS) were for instance describedin Brassica species. However, genetic determinants for male sterilitycan also be located in the nuclear genome. Male sterile plants can alsobe obtained by plant biotechnology methods such as genetic engineering.A particularly useful means of obtaining male-sterile plants isdescribed in WO 89/10396 in which, for example, a ribonuclease such asbarnase is selectively expressed in the tapetum cells in the stamens.Fertility can then be restored by expression in the tapetum cells of aribonuclease inhibitor such as barstar.

Plants or plant cultivars (obtained by plant biotechnology methods suchas genetic engineering) which may be treated according to the inventionare herbicide-tolerant plants, i.e., plants made tolerant to one or moregiven herbicides. Such plants can be obtained either by genetictransformation, or by selection of plants containing a mutationimparting such herbicide tolerance.

The following non-limiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Formula for the Efficacy of the Combination ofMultiple Active Ingredients

A synergistic effect of active ingredients is present when the activityof the active ingredient combinations exceeds the total of theactivities of the active ingredients when applied individually. Theexpected activity for a given combination of two active ingredients canbe calculated as follows (cf. Colby, S. R., “Calculating Synergistic andAntagonistic Responses of Herbicide Combinations,” Weeds 1967, 15,20-22):

If

-   -   X is the efficacy when active ingredient A is applied at an        application rate of m ppm (or g/ha),    -   Y is the efficacy when active ingredient B is applied at an        application rate of n ppm (or g/ha),

E is the efficacy when the active ingredients A and B are applied atapplication rates of m and n ppm (or g/ha), respectively, and

-   -   then

$E = {X + Y - \frac{X \cdot Y}{100}}$

If the actual activity exceeds the calculated value, then the activityof the combination is superadditive, i.e., a synergistic effect exists.In this case, the efficacy which was actually observed must be greaterthan the value for the expected efficacy (E) calculated from theabove-mentioned formula.

For instance, the formula and analysis can be applied to an evaluationof plant growth promotion. Such an assay is evaluated several days afterthe applications to plants. 100% means plant weight which corresponds tothat of the untreated control plant. Efficacy means in this case theadditional % of plant weight in comparison to that of the untreatedcontrol. For example, a treatment that resulted in plant weights thatwere 120% compared to the untreated control plant would have an efficacyof 20%. If the plant growth promotion effect for the combination (i.e.,the observed efficacy for % shoot weights of plants treated with thecombination) exceeds the calculated value, then the activity of thecombination is superadditive, i.e., a synergistic effect exists.

The formula and analysis can also be used to evaluate synergy in diseasecontrol assays. The degree of efficacy expressed in % is denoted. 0%means an efficacy which corresponds to that of the control while anefficacy of 100% means that no disease is observed.

If the actual insecticidal or fungicidal activity exceeds the calculatedvalue, then the activity of the combination is superadditive, i.e., asynergistic effect exists. In this case, the efficacy which is actuallyobserved must be greater than the value for the expected efficacy (E)calculated from the above-mentioned formula.

A further way of demonstrating a synergistic effect is the method ofTammes (cf. “Isoboles, A Graphic Representation of Synergism inPesticides,” in Neth. J. Plant Path., 1964, 70, 73-80).

Example 2 Plant Growth Promotion with Bacillus subtilis QST713 andRecombinant Bacillus thuringiensis Cells Expressing Phospholipase C

Experiments were conducted to analyze efficacy of a combination ofBacillus subtilis QST713-based product and a fermentation product ofrecombinant Bacillus thuringiensis cells expressing phospholipase C(“BEPC”). Maize seeds were grown in sterile mixture of synthetic mediaand sand in small three-inch square pots on light racks in a plantgrowth room at 25-28° C. and 50% humidity for about 14 days. Two seedswere planted in each pot. At planting, the growing media in each pot wasdrenched with the treatments described below. After 14 days, plants weremeasured for whole plant biomass. In some experiments, roots wereanalyzed using the WinRhizo Root scanner. In all tables throughout theseExamples, UTC refers to untreated control. “Calculated” refers to theexpected effect calculated using the above-described Colby equation and“Efficacy” refers to the actual effect observed.

The SERENADE® ASO product was diluted in water (1% and 5% volume tovolume) and the diluted solution was used to drench the growing media.The application rate of SERENADE® ASO refers to the amount of Bacillussubtilis QST713 (i.e., spore preparation) contained in the productSERENADE® ASO, which is 1.34%. The formulated product has a minimumspore concentration of 1×10⁹ cfu/g.

A recombinant Bacillus cereus family member (Bacillus thuringiensisBT013A) expressing phospholipase C on its exosporium (BEPC) wasgenerated as follows. To generate plasmids for expression of fusionproteins in Bacillus cereus family members, PCR fragments were generatedthat encoded the Bc1A promoter (SEQ ID NO: 85), a methionine startcodon, and amino acids 20-35 of Bc1A (SEQ ID NO: 1) followed by a sixalanine linker sequence fused in frame to Bacillus thuringiensis BT013Aphospholipase C (SEQ ID NO: 108). These PCR fragments were digested withXho1 and ligated into the Sall site of the pSUPER plasmid to generatethe plasmids pSUPER-Bc1A 20-35-Phospholipase. The pSUPER plasmid wasgenerated through fusion of the pUC57 plasmid (containing an ampicillinresistance cassette) with the pBC16-1 plasmid from Bacillus (containinga tetracycline resistance). This 5.5 kbp plasmid can replicate in bothE. coli and Bacillus spp. The pSUPER-Bc1A 20-35-Phospholipase plasmidswere transformed into and propagated in dam methylase negative E. colistrains and finally were transformed into Bacillus thuringiensis BT013A.

To obtain whole broth cultures of BEPC, 15 mL conicals containing brainheart infusion media (BHI) were inoculated with BEPC and grown for 7-8hours at around 30° C. at a shaker setting of 300 rpm. The next day, 250μl aliquots from each flask were inoculated into 250 mL flaskscontaining 50 mL of a yeast extract-based media and grown at about 30°C. After approximately 2 days of incubation, when sporulation was atleast 95% completed, the culture broth was harvested and colony formingunits calculated. The fermentation broth was diluted to 5% in 50 mLwater and the following colony forming units applied to each pot.

TABLE 3 Application Rate Whole PPM Plant and/or Biomass Found EfficacyCalculated Treatment CFU/pot (g) % % % UTC 3.23 100 SERENADE ® 703 4.71146 46 ASO 5% 2.62 × 10⁹ Bacillus 7 × 10⁸ 3.83 118 18 expressing CFU/potphospholipase C (BEPC) 5% SERENADE ® 2.62 × 10⁹ + 5.33 165 65 55.72 ASO5% + 7 × 10⁸ BEPC

The experiment was repeated as above but with a 1% dilution of theSERENADE® ASO product. Results are shown in Table 4, below.

TABLE 4 Whole Plant Application Biomass Found Efficacy CalculatedTreatment Rate (g) % % % UTC 3.22 100 SERENADE ® 140.7 PPM 3.62 112 12ASO 1% 5.24 × 10⁸ CFU/pot BEPC 7 × 10⁸ 3.76 117 17 CFU/pot SERENADE ®5.24 × 10⁸ 4.26 132 32 26.96 ASO 1% + CFU/pot + BEPC 7 × 10⁸ CFU/pot

Results of root volume measurements using the WinRhizo root scanner areshown in Table 5.

TABLE 5 Root Volume Average % Found % Efficacy % Calculated UTC 5.259100.0 0.0 SERENADE ® 6.282 119.5 19.5 ASO 1% BEPC 5% 6.249 118.8 18.8Ser 1% and 9.318 177.2 77.2 34.6 BEPC 5%

Results indicate a superadditive plant yield effect when combiningSERENADE® ASO and BEPC.

Example 3 Plant Growth Promotion with Bacillus subtilis QST713 andRecombinant Bacillus thuringiensis Cells Expressing Endoglucanase

Experiments similar to those in Example 2 were conducted using arecombinant Bacillus thuringiensis cells expressing endoglucanase (SEQID NO: 107), referred to in these examples as BEE. Whole broth culturesof BEE were created as described above, except that endoglucanase (SEQID NO: 107), rather than phosphpolipase, was used. Effects on plantyield of combinations of BEE with SERENADE® ASO are shown in the tablesbelow.

TABLE 6 Whole Plant Application Biomass Found Efficacy CalculatedTreatment Rate (g) % % % UTC 3.23 100 SERENADE ® 703 PPM 4.71 146 46 ASO5% 2.62 × 10⁹ CFU/pot BEE 5% 7.85 × 10⁸ 3.7 114 14 CFU/pot SERENADE ®2.62 × 10⁹ 5.89 182 82 53.56 ASO 5% + CFU/pot + BEE 5% 7.85 × 10⁸CFU/pot

TABLE 7 Whole Plant Application Biomass Found Efficacy CalculatedTreatment Rate (g) % % % UTC 3.22 100 SERENADE ® 140.7 PPM 3.62 112 12ASO 1% 5.24 × 10⁸ CFU/pot BEE 5% 7.85 × 10⁸ 3.92 121 21 CFU/potSERENADE ® 5.24 × 10⁸ 4.40 136 36 30.48 ASO 1% + CFU/pot + BEE 5% 7.85 ×10⁸ CFU/pot

TABLE 8 Root Volume Average % Found % Efficacy % Calculated UTC 5.259100.0 0.0 SERENADE ® 6.282 119.5 19.5 ASO 1% BEE 5% 6.704 127.5 27.5 Ser1% and 8.209 156.1 56.1 41.6 BEE 5%

The above results show a superadditive effect on plant yield whenBacillus subtilis QST713 and BEE are applied in combination.

Example 4 Plant Growth Promotion with Bacillus firmus strain I-1582 andRecombinant Bacillus thuringiensis Cells

Maize seeds will be grown in loamy sand in the greenhouse at 20° C. and70% humidity for about 11 days. After about 11 days from the time oftreatment the seedlings will be cut off above the soil and the freshweight will be determined.

Recombinant Bacillus thuringiensis cells expressing an endoglucanaseencoded by SEQ ID NO: 107 or a phospholipase C encoded by SEQ ID NO: 108and prepared as described above will be applied at about 50 μg/kernel.Bacillus firmus strain I-1582 will also be applied at about 50μg/kernel.

It is expected that the maize plants treated with the recombinantBacillus thuringiensis in combination with the Bacillus firmus strainI-1582 will have % shoot weights that exceed the calculated value basedon the % shoot weights from the maize plants treated with the two activeingredients alone, i.e., a synergistic effect will be observed.

1. A composition comprising: a) recombinant exosporium-producingBacillus cells that express a fusion protein comprising: (i) at leastone plant growth stimulating protein or peptide; and (ii) a targetingsequence, exosporium protein, or exosporium protein fragment; and b) aBacillus firmus strain in a synergistically effective amount.
 2. Thecomposition of claim 1, wherein the at least one plant growthstimulating protein or peptide is selected from the group consisting ofan enzyme involved in the production or activation of a plant growthstimulating compound and an enzyme that degrades or modifies abacterial, fungal, or plant nutrient source.
 3. The composition of claim1, wherein the exosporium-producing Bacillus cells are cells of aBacillus cereus family member.
 4. The composition according of claim 3,wherein the Bacillus cereus family member is selected from the groupconsisting of Bacillus anthracis, Bacillus cereus, Bacillusthuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillussamanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillustoyoiensis and combinations thereof.
 5. The composition according toclaim 1, wherein the targeting sequence or exosporium protein comprises:an amino acid sequence having at least about 43% identity with aminoacids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35is at least about 54%; a targeting sequence comprising amino acids 1-35of SEQ ID NO: 1; a targeting sequence comprising amino acids 20-35 ofSEQ ID NO: 1; a targeting sequence comprising amino acids 22-31 of SEQID NO: 1; a targeting sequence comprising amino acids 22-33 of SEQ IDNO: 1; a targeting sequence comprising amino acids 20-31 of SEQ ID NO:1; a targeting sequence comprising SEQ ID NO: 1; or an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO:
 2. 6. The composition according to claim 2, wherein theenzyme involved in the production or activation of a plant growthstimulating compound is selected from the group consisting of an acetoinreductase, an indole-3-acetamide hydrolase, a tryptophan monooxygenase,an acetolactate synthetase, an α-acetolactate decarboxylase, a pyruvatedecarboxylase, a diacetyl reductase, a butanediol dehydrogenase, anaminotransferase, a tryptophan decarboxylase, an amine oxidase, anindole-3-pyruvate decarboxylase, an indole-3-acetaldehyde dehydrogenase,a tryptophan side chain oxidase, a nitrile hydrolase, a nitrilase, apeptidase, a protease, an adenosine phosphate isopentenyltransferase, aphosphatase, an adenosine kinase, an adenine phosphoribosyltransferase,CYP735A, a 5′ribonucleotide phosphohydrolase, an adenosine nucleosidase,a zeatin cis-trans isomerase, a zeatin O-glucosyltransferase, aβ-glucosidase, a cis-hydroxylase, a CK cis-hydroxylase, a CKN-glucosyltransferase, a 2,5-ribonucleotide phosphohydrolase, anadenosine nucleosidase, a purine nucleoside phosphorylase, a zeatinreductase, a hydroxylamine reductase, a 2-oxoglutarate dioxygenase, agibberellic 2B/3B hydrolase, a gibberellin 3-oxidase, a gibberellin20-oxidase, a chitosanase, a chitinase, a β-1,3-glucanase, aβ-1,4-glucanase, a β-1,6-glucanase, an aminocyclopropane-1-carboxylicacid deaminase, and an enzyme involved in producing a nod factor.
 7. Thecomposition of claim 6, wherein the enzyme involved in the production oractivation of a plant growth stimulating compound is a chitosanase. 8.The composition of claim 7, wherein the fusion protein comprises SEQ IDNO:
 109. 9. The composition according to claim 2, wherein the enzymethat degrades or modifies a bacterial, fungal, or plant nutrient sourceis selected from the group consisting of a cellulase, a lipase, a ligninoxidase, a protease, a glycoside hydrolase, a phosphatase, anitrogenase, a nuclease, an amidase, a nitrate reductase, a nitritereductase, an amylase, an ammonia oxidase, a ligninase, a glucosidase, aphospholipase, a phytase, a pectinase, a glucanase, a sulfatase, aurease, a xylanase, and a siderophore.
 10. The composition of claim 9,wherein the enzyme is a cellulase selected from the group consisting ofan endocellulase, an exocellulase, and a β-glucosidase.
 11. Thecomposition of claim 10, wherein the fusion protein comprises a Bacillussubtilis endoglucanase.
 12. The composition of claim 11, wherein thefusion protein comprises SEQ ID NO:
 107. 13. The composition of claim12, wherein the recombinant Bacillus cells are derived from Bacillusthuringiensis BT013A.
 14. The composition of claim 9, wherein the enzymeis a phospholipase.
 15. The composition of claim 14, wherein the fusionprotein comprises SEQ ID NO:
 108. 16. The composition according to claim1, wherein the fusion protein is expressed under the control of asporulation promoter native to the targeting sequence, exosporiumprotein or exosporium protein fragment of the fusion protein. 17-21.(canceled)
 22. The composition according to claim 1, wherein the atleast one biological control agent is Bacillus firmus strain I-1582. 23.A seed treated with the composition according to claim
 1. 24-25.(canceled)
 26. A method of treating a plant, a plant part, or the locussurrounding the plant to enhance plant growth and/or promote planthealth comprising the step of simultaneously or sequentially applying:a) recombinant exosporium-producing Bacillus cells that express a fusionprotein comprising: (i) at least one plant growth stimulating protein orpeptide; and (ii) a targeting sequence, exosporium protein, orexosporium protein fragment; and b) a Bacillus firmus strain in asynergistically effective amount.
 27. The method according to claim 26,wherein the targeting sequence or exosporium protein comprises: an aminoacid sequence having at least about 43% identity with amino acids 20-35of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at leastabout 54%; a targeting sequence comprising amino acids 1-35 of SEQ IDNO: 1; a targeting sequence comprising amino acids 20-35 of SEQ ID NO:1; a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1; atargeting sequence comprising amino acids 22-33 of SEQ ID NO: 1; atargeting sequence comprising amino acids 20-31 of SEQ ID NO: 1; atargeting sequence comprising SEQ ID NO: 1; or an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO:
 2. 28-31. (canceled)